Activated polyoxazolines and conjugates and compositions comprising the same

ABSTRACT

The present disclosure provides POZ derivatives having a range of active functional groups allowing conjugation of POZ derivatives to a variety of target molecules under a wide range of reaction conditions to produce a hydrolytically stable target molecule-POZ conjugate. Furthermore, the present disclosure provides novel methods of synthesis for the disclosed POZ derivatives and hydrolytically stable target molecule-POZ conjugates created using the disclosed terminally activated monofunctional POZ derivatives. In one embodiment, the POZ derivative is a terminally activated monofunctional POZ derivative.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No.15/453,686, filed Mar. 8, 2017 (currently pending). U.S. applicationSer. No. 15/453,686 is a continuation of U.S. application Ser. No.14/663,863, filed Mar. 20, 2015 (currently abandoned). U.S. applicationSer. No. 14/663,863 is a continuation of U.S. application Ser. No.13/961,576, filed Aug. 7, 2013 (currently abandoned). U.S. applicationSer. No. 13/961,576 is a continuation of U.S. application Ser. No.13/676,048, filed Nov. 13, 2012 (currently abandoned). U.S. applicationSer. No. 13/676,048 is a continuation of U.S. application Ser. No.13/276,910, filed Oct. 19, 2011 (currently abandoned). U.S. applicationSer. No. 13/276,910 is a continuation of U.S. application Ser. No.12/622,264, filed Nov. 19, 2009 (currently abandoned). U.S. applicationSer. No. 12/622,264 claims the benefit of U.S. Provisional ApplicationNos. 61/116,246, filed Nov. 19, 2008 (currently expired), and61/116,252, filed Nov. 19, 2008 (currently expired), and is acontinuation in part of U.S. application Ser. No. 12/529,001, filed Aug.27, 2009, now U.S. Pat. No. 7,943,141, issued May 17, 2011. U.S. Pat.No. 7,943,141 is a 371 of International Application No.PCT/US2008/002626, filed Feb. 28, 2008, (currently expired), whichclaims the benefit of U.S. Provisional Application No. 60/892,212, filedFeb. 28, 2007 (currently expired).

FIELD OF THE DISCLOSURE

The present disclosure relates to polyoxazoline polymers, polyoxazolinederivatives, methods of synthesis and intermediate compounds useful inproducing the foregoing, and target molecule-polyoxazoline conjugatesproduced using such polyoxazoline derivatives. In one embodiment, thepresent disclosure describes target molecule-polyoxazoline conjugateswhere the target molecule is a of protein or peptide, methods ofsynthesis for such target molecule-polyoxazoline conjugates, includingenzymatic methods, and intermediate compounds useful in or produced insuch methods and the biological activities of the produced polyoxazolineconjugates. The methods of the present disclosure allow for thepreparation of polyoxazoline conjugates having high purity andhomogeneity.

BACKGROUND

Polymer-modified therapeutics have proven to be of great utility inmodern pharmaceutical science. In particular, proteins coupled topolyethylene glycols (PEGs) now constitute a number of therapeutics ofgreat importance for treatment of a range of diseases. Due to thesuccess of polymer-modified therapeutics, it is of interest to expandthe range of polymers having such applications, especially to providepolymers having properties not possessed by polyethylene glycol.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates the living-cation mechanism for 2-alkyl-2-oxazoline(e.g., 2-ethyl-2-oxazoline) polymerization where —OTf is —OSO₂-CF₃ or“triflate” and Nuc⁻ is a negative nucleophile.

FIG. 2 shows a mechanism for the elimination-dimerization mechanism forchain transfer during polymerization of polyoxazoline derivatives,illustrated here as 2-ethyl-2-oxazoline.

FIG. 3 shows the amino acid sequence of human recombinanterythropoietin.

FIG. 4 shows the effect of GCSF and 10 kDa H-PEOZ-N^(ter)-GCSF, 20 kDaH-PEOZ-N^(ter)-GCSF and 20 kDa H-PEOZ-GCSF conjugates on NSF-60 cellproliferation.

FIG. 5 shows blood neutrophil counts in male Sprague-Dawley ratsfollowing intravenous injection of GCSF and 10 kDa PEOZ-N^(ter)-GCSF, 20kDa PEOZ-N^(ter)-GCSF, 20 kDa PEOZ-hex-GCSF conjugates.

FIG. 6 shows the formation of a 5 kDa POZ-ethylenediamine-apoMBconjugate.

FIG. 7 shows a chromatograms of the peptide maps of Endoprotease Lys Cdigested EPO. FIG. 8 shows a chromatograms of the peptide maps ofEndoprotease Lys C digested PEOZ-EPO conjugate.

FIG. 9 shows the effect of PEOZ-EPO conjugates by UT-7 cellproliferation.

FIG. 10 shows the concentrations of EPO and a PEOZ-EPO conjugate in ratserum.

FIG. 11 shows the effect of EPO and PEOZ-EPO conjugates on thereticulocyte counts in rat blood.

DETAILED DESCRIPTION Definitions

As used herein, the term “POZ”, “POZ compound” or “POZ polymer” refersto a polymer of 2-substituted-2-oxazoline containing a repeating unithaving the structure—[N(COR₇)CH₂CH₂]_(n)— in which R₇ is independentlyselected for each repeating unit from an unsubstituted or substitutedalkyl, alkenyl, aralkyl or heterocyclylalkyl group and n is from 3-1000;in one embodiment, the unsubstituted or substituted alkyl, alkenyl,aralkyl or heterocyclylalkyl groups comprise, from 1-10 carbon atoms, ina further specific embodiment, R₇ is methyl, ethyl or n-propyl.

As used herein, the term “PMOZ” refers to POZ with the repeating unithaving the structure —[N(COCH₃)CH₂CH₂]_(n)—.

As used herein, the term “PEOZ” refers to POZ with the repeating unithaving the structure —[N(COCH₂CH₃)CH₂CH₂]_(n)—.

As used herein, the term M- when used in conjunction with a POZ polymeror derivative, such as M-POZ, M-PMOZ or M-PEOZ, indicates the nitrogenon the initiating end is bound to methyl. Likewise, the term H- whenused in conjunction with a POZ polymer or derivative, such as H-POZ,H-PMOZ or H-PEOZ, indicates the nitrogen on the initiating end is boundto hydrogen.

As used herein, the term “POZ derivative” or “polyoxazoline derivative”refers to a structure comprising a POZ polymer, the POZ polymer having asingle active functional group on the terminal end of the POZ polymer,the functional group capable of forming a linkage, directly orindirectly, with a chemical group on a target molecule; in oneembodiment the POZ derivative is a monofunctional POZ derivative.

As used herein, the term “target molecule” refers to any molecule havingtherapeutic, diagnostic application or a targeting function, wherein thetarget molecule is capable of reacting with an active functional groupon a POZ polymer or a POZ derivative of the present disclosure,including, but not limited to, a therapeutic moiety (such as but notlimited to a drug), a diagnostic moiety, a targeting moiety, an organicsmall molecule, a lipid, an oligonucleotide, a polypeptide, an antibody,an antibody fragment and a protein. Exemplary target molecules include,but are not limited to, lipids, erythropoietin, granulocyte colonystimulating factor, dynorphin A. lysozyme, human growth hormone,apomyoglobin and hyaluronic acid.

As used herein, the term “hydrolytically stable target molecule-POZconjugate” refers to a conjugate of a POZ derivative of the presentdisclosure and a target molecule, such that all the chemical linkagesbetween the POZ conjugate and the target molecule are hydrolyticallystable.

As used herein, the term “hydrolytically stable” refers to a linkagethat is stable in aqueous solutions under physiological conditions; inone embodiment, such linkages are stable for at least 12 hours, 24hours, 48 hours, 96 hours, 192 hours or greater; in an alternateembodiment such linkages are stable indefinitely.

As used herein, the term “hydrolytically unstable” refers to a linkagethat is not stable in aqueous solutions under physiological conditions.

As used herein, the term “physiological conditions” refers to an aqueoussolution having a pH from 6-8 and a temperature from 30-42 degreesCelsius.

As used herein, the term “active functional group” refers to thosegroups that react readily with electrophilic or nucleophilic groups orthat react readily by cylcoaddition reactions, in contrast to thosegroups that require strong catalysis, high temperatures or impracticalreaction conditions in order to react.

As used herein, the term “link”, “linked” “linkage” or “linker” whenused with respect to a POZ derivative described herein, or componentsthereof, refers to groups or bonds that normally are formed as theresult of a chemical reaction and typically are covalent linkages.

As used herein, the term “protected” with respect to hydroxyl groups,amine groups, sulfhydryl groups and other reactive groups refers toforms of these functionalities which are protected from undesirablereaction with a protecting group known to those skilled in the art suchas those set forth in Protective Groups in Organic Synthesis, Greene, T.W.; Wuts, P. G. M., John Wiley & Sons, New York, N.Y., (3rd Edition,1999) which can be added or removed using the procedures set forththerein. Examples of protected hydroxyl groups include, but are notlimited to, silyl ethers such as those obtained by reaction of ahydroxyl group with a reagent such as, but not limited to,t-butyldimethyl-chlorosilane, trimethylchlorosilane,triisopropylchlorosilane, triethylchlorosilane; substituted methyl andethyl ethers such as, but not limited to methoxymethyl ether,methythiomethyl ether, benzyloxymethyl ether, t-butoxymethyl ether,2-methoxyethoxymethyl ether, tetrahydropyranyl ethers, 1-ethoxyethylether, allyl ether, benzyl ether; esters such as, but not limited to,benzoylformate, formate, acetate, trichloroacetate, and trifluoracetate.Examples of protected amine groups include, but are not limited to,amides such as, formamide, acetamide, trifluoroacetamide, and benzamide;imides, such as phthalimide, and dithiosuccinimide; and others. Examplesof protected sulfhydryl groups include, but are not limited to,thioethers such as S-benzyl thioether, and S-4-picolyl thioether;substituted S-methyl derivatives such as hemithio, dithio and aminothioacetals; and others.

As used herein, the term “alkyl”, whether used alone or as part of asubstituent group, includes straight hydrocarbon groups comprising fromone to twenty carbon atoms. Thus the phrase includes straight chainalkyl groups such as methyl, ethyl, propyl, butyl, pentyl, hexyl,heptyl, octyl, nonyl, decyl, undecyl, dodecyl and the like. The phrasealso includes branched chain isomers of straight chain alkyl groups,including but not limited to, the following which are provided by way ofexample: —CH(CH₃)₂, —CH(CH₃)(CH₂CH₃), —CH(CH₂CH₃)₂, —C(CH₃)₃,—C(CH₂CH₃)₃, —CH₂ CH(CH₃)₂, —CH₂CH(CH₃)(CH₂CH₃), —CH₂CH(CH₂CH₃)₂,—CH₂C(CH₃)₃, —CH₂C(CH₂CH₃)₃, —CH(CH₃)CH(CH₃)(CH₂CH₃), CH₂CH₂CH(CH₃)₂,—CH₂CH₂CH(CH₃)(CH₂CH₃), —CH₂CH₂CH(CH₂CH₃)₂, —CH₂CH₂C(CH₃)₃,—CH₂CH₂C(CH₂CH₃)₃, —CH(CH₃)CH₂CH(CH₃)₂, —CH(CH₃)CH(CH₃)CH(CH₃)CH(CH₃)₂,—CH(CH₂ CH₃)CH(CH₃)CH(CH₃)(CH₂CH₃), and others. The phrase also includescyclic alkyl groups such as cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, cycloheptyl, and cyclooctyl and such rings substituted withstraight and branched chain alkyl groups as defined above. The phrasealso includes polycyclic alkyl groups such as, but not limited to,adamantyl norbornyl, and bicyclo[2.2.2] octyl and such rings substitutedwith straight and branched chain alkyl groups as defined above.

As used herein, the term “alkenyl”, whether used alone or as part of asubstituent group, includes an alkyl group having at least one doublebond between any two adjacent carbon atoms.

As used herein, the term “alkynyl”, whether used alone or as part of asubstituent group, includes an alkyl group having at least one triplebond between any two adjacent carbon atoms.

As used herein, the term “unsubstituted alkyl”, “unsubstituted alkenyl”and “unsubstituted alkynyl” refers to alkyl, alkenyl and alkynyl groupsthat do not contain heteroatoms.

The phrase “substituted alkyl”, “substituted alkenyl” and “unsubstitutedalkynyl” refers to alkyl alkenyl and alkynyl groups as defined above inwhich one or more bonds to a carbon(s) or hydrogen(s) are replaced by abond to non-hydrogen or non-carbon atoms such as, but not limited to, anoxygen atom in groups such as alkoxy groups and aryloxy groups; a sulfuratom in groups such as, alkyl and aryl sulfide groups, sulfone groups,sulfonyl groups, and sulfoxide groups; a silicon atom in groups such asin trialkylsilyl groups, dialkylarylsilyl groups, alkyldiarylsilylgroups, and triarylsilyl groups; and other heteroatoms in various othergroups.

As used herein, the term “unsubstituted aryl” refers to monocyclic orbicyclic aromatic hydrocarbon groups having 6 to 12 carbon atoms in thering portion, such as, but not limited to, phenyl, naphthyl,anthracenyl, biphenyl and diphenyl groups, that do not containheteroatoms. Although the phrase “unsubstituted aryl” includes groupscontaining condensed rings such as naphthalene, it does not include arylgroups that have other groups such as alkyl or halo groups bonded to oneof the ring members, as aryl groups such as tolyl are considered hereinto be substituted aryl groups as described below. Unsubstituted arylgroups may be bonded to one or more carbon atom(s), oxygen atom(s),nitrogen atom(s), and/or sulfur atom(s) in the parent compound, however.

As used herein, the term “substituted aryl group” has the same meaningwith respect to unsubstituted aryl groups that substituted alkyl groupshad with respect to unsubstituted alkyl groups. However, a substitutedaryl group also includes aryl groups in which one of the aromaticcarbons is bonded to one of the non-carbon or non-hydrogen atoms, suchas, but not limited to, those atoms described above with respect to asubstituted alkyl, and also includes aryl groups in which one or morearomatic carbons of the aryl group is bonded to a substituted and/orunsubstituted alkyl, alkenyl, or alkynyl group as defined herein. Thisincludes bonding arrangements in which two carbon atoms of an aryl groupare bonded to two atoms of an alkyl or alkenyl, group to define a fusedring system (e.g. dihydronaphthyl or tetrahydronaphthyl). Thus, thephrase “substituted aryl” includes, but is not limited to tolyl, andhydroxyphenyl among others.

As used herein, the term “unsubstituted aralkyl” refers to unsubstitutedalkyl or alkenyl groups as defined above in which a hydrogen or carbonbond of the unsubstituted or substituted alkyl or alkenyl group isreplaced with a bond to a substituted or unsubstituted aryl group asdefined above. For example, methyl (CH₃) is an unsubstituted alkylgroup. If a hydrogen atom of the methyl group is replaced by a bond to aphenyl group, such as if the carbon of the methyl were bonded to acarbon of benzene, then the compound is an unsubstituted aralkyl group(i.e., a benzyl group).

As used herein, the term “substituted aralkyl” has the same meaning withrespect to unsubstituted aralkyl groups that substituted aryl groups hadwith respect to unsubstituted aryl groups. However, a substitutedaralkyl group also includes groups in which a carbon or hydrogen bond ofthe alkyl part of the group is replaced by a bond to a non-carbon or anon-hydrogen atom.

As used herein, the term “unsubstituted heterocyclyl” refers to botharomatic and nonaromatic ring compounds including monocyclic, bicyclic,and polycyclic ring compounds such as, but not limited to, quinuclidyl,containing 3 or more ring members of which one or more is a heteroatomsuch as, but not limited to, N, O, and S. Although the phrase“unsubstituted heterocyclyl” includes condensed heterocyclic rings suchas benzimidazolyl, it does not include heterocyclyl groups that haveother groups such as alkyl or halo groups bonded to one of the ringmembers, as compounds such as 2-methylbenzimidazolyl are “substitutedheterocyclyl” groups as defined below. Examples of heterocyclyl groupsinclude, but are not limited to: unsaturated 3 to 8 membered ringscontaining 1 to 4 nitrogen atoms such as, but not limited to pyrrolyl,pyrrolinyl, imidazolyl, pyrazolyl, pyridyl, dihydropyridyl, pyrimidyl,pyrazinyl, pyridazinyl, triazolyl, tetrazolyl; saturated 3 to 8 memberedrings containing 1 to 4 nitrogen atoms such as, but not limited to,pyrrolidinyl, imidazolidinyl, piperidinyl, piperazinyl; condensedunsaturated heterocyclic groups containing 1 to 4 nitrogen atoms suchas, but not limited to, indolyl, isoindolyl, indolinyl, indolizinyl,benzimidazolyl, quinolyl, isoquinolyl, indazolyl, benzotriazolyl;unsaturated 3 to 8 membered rings containing 1 to 2 oxygen atoms and 1to 3 nitrogen atoms such as, but not limited to, oxazolyl, isoxazolyl,oxadiazolyl; saturated 3 to 8 membered rings containing 1 to 2 oxygenatoms and 1 to 3 nitrogen atoms such as, but not limited to,morpholinyl; unsaturated condensed heterocyclic groups containing 1 to 2oxygen atoms and 1 to 3 nitrogen atoms, for example, benzoxazolyl,benzoxadiazolyl, benzoxazinyl (e.g. 2H-1,4-benzoxazinyl etc.);unsaturated 3 to 8 membered rings containing 1 to 3 sulfur atoms and 1to 3 nitrogen atoms such as, but not limited to, thiazolyl,isothiazolyl, thiadiazolyl (e.g. 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl,1,3,4-thiadiazolyl, 1,2,5-thiadiazolyl, etc.); saturated 3 to 8 memberedrings containing 1 to 2 sulfur atoms and 1 to 3 nitrogen atoms such as,but not limited to, thiazolodinyl; saturated and unsaturated 3 to 8membered rings containing 1 to 2 sulfur atoms such as, but not limitedto, thienyl, dihydrodithiinyl, dihydrodithionyl, tetrahydrothiophene,tetrahydrothiopyran; unsaturated condensed heterocyclic rings containing1 to 2 sulfur atoms and 1 to 3 nitrogen atoms such as, but not limitedto, benzothiazolyl, benzothiadiazolyl, benzothiazinyl (e.g.2H-1,4-benzothiazinyl, etc.), dihydrobenzothiazinyl (e.g.2H-3,4-dihydrobenzothiazinyl, etc.), unsaturated 3 to 8 membered ringscontaining oxygen atoms such as, but not limited to furyl; unsaturatedcondensed heterocyclic rings containing 1 to 2 oxygen atoms such asbenzodioxolyl (e.g. 1,3-benzodioxoyl, etc.); unsaturated 3 to 8 memberedrings containing an oxygen atom and 1 to 2 sulfur atoms such as, but notlimited to, dihydrooxathiinyl; saturated 3 to 8 membered ringscontaining 1 to 2 oxygen atoms and 1 to 2 sulfur atoms such as1,4-oxathiane; unsaturated condensed rings containing 1 to 2 sulfuratoms such as benzothienyl, benzodithiinyl; and unsaturated condensedheterocyclic rings containing an oxygen atom and 1 to 2 oxygen atomssuch as benzoxathiinyl. Heterocyclyl group also include those describedabove in which one or more S atoms in the ring is double-bonded to oneor two oxygen atoms (sulfoxides and sulfones). For example, heterocyclylgroups include tetrahydrothiophene, tetrahydrothiophene oxide, andtetrahydrothiophene 1,1-dioxide. Preferred heterocyclyl groups contain 5or 6 ring members. More preferred heterocyclyl groups includemorpholine, piperazine, piperidine, pyrrolidine, imidazole, pyrazole,1,2,3-triazole, 1,2,4-triazole, tetrazole, thiomorpholine,thiomorpholine in which the S atom of the thiomorpholine is bonded toone or more O atoms, pyrrole, homopiperazine, oxazolidin-2-one,pyrrolidin-2-one, oxazole, quinuclidine, thiazole, isoxazole, furan, andtetrahydrofuran.

As used herein, the term “substituted heterocyclyl” has the same meaningwith respect to unsubstituted heterocyclyl groups that substituted alkylgroups had with respect to unsubstituted alkyl groups. However, asubstituted heterocyclyl group also includes heterocyclyl groups inwhich one of the carbons is bonded to one of the non-carbon ornon-hydrogen atom, such as, but not limited to, those atoms describedabove with respect to a substituted alky and substituted aryl groups andalso includes heterocyclyl groups in which one or more carbons of theheterocyclyl group is bonded to a substituted and/or unsubstitutedalkyl, alkenyl or aryl group as defined herein. This includes bondingarrangements in which two carbon atoms of an heterocyclyl group arebonded to two atoms of an alkyl, alkenyl, or alkynyl group to define afused ring system. Examples, include, but are not limited to,2-methylbenzimidazolyl, 5-methylbenzimidazolyl, 5-chlorobenzthiazolyl,1-methyl piperazinyl, and 2-chloropyridyl among others.

As used herein, the term “unsubstituted heterocyclylalkyl” refers tounsubstituted alkyl or alkenyl groups as defined above in which ahydrogen or carbon bond of the unsubstituted alkyl or alkenyl group isreplaced with a bond to a substituted or unsubstituted heterocyclylgroup as defined above. For example, methyl (CH₃) is an unsubstitutedalkyl group. If a hydrogen atom of the methyl group is replaced by abond to a heterocyclyl group, such as if the carbon of the methyl werebonded to carbon 2 of pyridine (one of the carbons bonded to the N ofthe pyridine) or carbons 3 or 4 of the pyridine, then the compound is anunsubstituted heterocyclylalkyl group.

As used herein, the term “substituted heterocyclylalkyl” has the samemeaning with respect to unsubstituted heterocyclylalkyl groups thatsubstituted aryl groups had with respect to unsubstituted aryl groups.However, a substituted heterocyclylalkyl group also includes groups inwhich a non-hydrogen atom is bonded to a heteroatom in the heterocyclylgroup of the heterocyclylalkyl group such as, but not limited to, anitrogen atom in the piperidine ring of a piperidinylalkyl group.

General Description

Polyoxazolines (POZ) are polymers prepared from2-substituted-2-oxazoline monomers. These polymers are water soluble andhave been reported to be nontoxic in mammalian model systems. POZ isgenerally prepared by reaction of the appropriate stoichiometric amountof 2-alkyl-2-oxazoline with an electrophilic initiator, such as methylp-toluenesulfonate (or “tosylate”, CH₃—OSO₂—C₆H₄—CH₃) or methyl triflate(CH₃—OSO₂—CF₃), followed by termination with a nucleophile such ashydroxide or an amine. The polymer produced is conveniently described inshorthand with the initiating group designated by the leftmost group andthe terminating group designated by the rightmost group, with the2-alkyl-2-oxazoline component in the middle. Therefore, when thisshorthand description is used in the current specification, it isintended that the left side of the designation presents the “initiatorend” and the right side of the designation presents the “terminal end”,unless designated otherwise.

For example, when the 2-substituted-2-oxazoline is 2-methyl-2-oxazoline,methyl tosylate is used as the initiator and hydroxide is used as theterminator, the following polymer is produced:

CH₃—[N(COCH₃)CH₂CH₂]_(n)—OH

The polymer above is conveniently described in shorthand notation asM-PMOZ-OH, in which the methyl initiator is designated by the leftmost M(at the initiator end), PMOZ represents polymethyloxazoline with themethyl of the repeating unit designated by the M of PMOZ, and theterminating hydroxyl is designated by the —OH (at the terminal end). Thedegree of polymerization, n, can range from approximately 3 to about1000.

Another commonly used monomer is 2-ethyl-2-oxazoline, which with methyltriflate initiation and hydroxide termination would provide thefollowing POZ polymer:

CH₃—[N(COCH₂CH₃)CH₂CH₂]_(n)—OH

The polymer above is conveniently described in shorthand notation asM-PEOZ-OH, in which the methyl initiator is designated by the leftmost M(at the initiator end), PEOZ represents polymethyloxazoline with theethyl of the repeating unit designated by the E of PEOZ, and theterminating hydroxyl is designated by the —OH (at the terminal end).

More complex electrophiles and nucleophiles can be used. For example,initiation of 2-ethyl-2-oxazoline polymerization with benzyl bromide andtermination with excess ethylene diamine yields the following polymer:

C₆H₅—CH₂—[N(COCH₂CH₃)CH₂CH₂]_(n)—NH—CH₂CH₂—NH₂

Also, different monomers can be used in the same polymer to yieldvarious random and block copolymers.

The polymerization process is referred to as a living, cationicpolymerization since initiation with an electrophile produces anoxazolinium cation that then reacts in a chain reaction with additionalmonomer units to produce a growing, “living” cation.

One can predict the products of termination by assuming that the livingcation can be represented in the following non-cyclic form, although inreality the cyclic form is certainly the most important, and the desiredproducts are produced by nucleophilic attack on the 5-position of thering:

CH₃—[N(COCH₃)CH₂CH₂]_(n)—N(COCH₃)CH₂CH₂ ⁺

In the current disclosure this cation will be represented as M-PMOZ⁺. Asnoted above, this POZ cation can be “terminated” by reacting withnucleophiles such as hydroxide or amines. Interestingly, terminationwith the weak nucleophile water does not give the desired product of5-attack (the “thermodynamic” product) but rather gives attack in the2-position (the “kinetic” product). This kinetic product is not stableand can rearrange to give an ester product or undergo reversal to cation(O. Nuyken, G. Maier, A. Gross, Macromol. Chem. Phys. 197, 83-85(1996)).

Hydroxyl terminated polymers can be further modified to give desiredderivatives. For example, Zalipsky reacted the terminal —OH withglutaric anhydride to give a POZ terminated with a glutarate group (M.C. Woodle, C. M. Engbers and S. Zalipsky, Bioconjugate Chem., 1994, 5,493-496).

M-PMOZ-O₂C—CH₂CH₂CH₂—CO₂H

The above polymer was activated as the succinimidyl ester and coupled tophospholipids and used to prepared POZ-modified liposomes. Theseliposomes were found to have similar properties to PEG-modifiedliposomes.

Amine terminated polymers also provide useful reactive groups forfurther derivatization. For example, termination with methyl amine givesa POZ terminated with the active group —NHCH₃. Termination with thecyclic diamine piperazine can also be useful.

Oxazoline polymerizations can also be initiated with functionalnucleophiles. For example, the electrophilic initiator ethyl3-bromopropionate has been used to initiate 2-ethyl-2-oxazolinepolymerization. Termination with hydroxide gives the following polymer:

HO₂C—CH₂CH₂—[N(COCH₂CH₃)CH₂CH₂]_(n)—OH

It is noteworthy that POZs having the same functional group on theinitiator end and the terminal end are chemically different because thegroup at the initiator end is attached to nitrogen while the group atthe terminal end is attached to carbon. For example, the following twopolymers are both propionic acid derivatives of PMOZ but differ in thatthe propionic acid at the initiator end is attached to nitrogen and thepropionic acid at the terminal end is attached to carbon (the beginningor ending monomer unit is shown for clarity):

HOOCCH₂CH₂—N(COCH₃)CH₂CH₂—PMOZ-OH

M-PMOZ-N(COCH₃)CH₂CH₂—O—CH₂CH₂COOH

Yet another route to preparing polyoxazolines with active functionalgroups is to copolymerize a monomer such as 2-ethyl-2-oxazoline with anoxazoline monomer having an active functional group in the 2-position.For example, Jordan and colleagues have prepared oxazolines withacetylenes and protected aldehydes, carboxylic acids and amines in the2-position (F. C. Gaertner, R. Luxenhofer, B. Blechert, R. Jordan and M.Essler, J. Controlled Release, 2007, 119, 291-300). Copolymerization ofthese functional monomers with 2-ethyl-2-oxazoline gives randomcopolymers with multiple pendent or side-chain active functional groups.For example, initiation with methyl triflate of polymerization of2-ethyl-2-oxazoline and 2-pentynyl-2-oxazoline, followed by terminationwith piperazine (NHC₄H₈NH) gives the following random copolymer:

CH₃—{[N(COCH₂CH₃)CH₂CH₂]_(n)—[N(COCH₂CH₂CH₂—CCH)CH₂CH₂]_(m)}_(ran)—NC₄H₈NH

The subscript “ran” indicates that the polymer is a random copolymer.Values of n are typically around 20-30 while m is around 2-5.

To couple a POZ to a target molecule, such as, but not limited to, apolypeptide, it is necessary to “activate” the polymer by attaching anactive functional group to at least one terminus of the polymer that iscapable of forming a linkage with a group on the target molecule. Therehas been little work done on activation of POZ for coupling to targetmolecules. The active group may be added at the initiator (left) end orterminal (right) end, or both. For example, when the target molecule isa polypeptide, the polypeptide has a number of amino groups on thesurface that can react with the active functional group on the POZ, andin the only published example of attachment of POZ to a protein, Myamotoand colleagues attached the POZ below to amino groups of the enzymecatalase (M. Myamoto, T. Saegusa, et al., Macromolecules, 1990, 23,3201-3205):

M-PMOZ-O₂C—CH₂CH₂CH₂—CO₂—NHS

In this case M-PMOZ-OH was reacted with glutaric anhydride, and theresulting carboxylic acid was activated with N-hydroxysuccinimide, whichis represented as NHS. NHS active esters are commonly used active formsof carboxylic acids. In this example the POZ-catalase conjugate provedto be active.

The prior art has described three terminally-activated POZ compoundscapable of being conjugated to a target molecule. However, each of thepreviously described POZ derivatives suffers from limitations. Zalipskydescribed an activated propionic acid POZ compound,NHS—OOCCH₂CH₂-PEOZ-OH where the activating group was attached to theinitiator end of the molecule (S. Zalipsky, C. B. Hansen, J. M. Oaks andT. M. Allen, J. Pharm. Sci., 85, 133-137 (1996)). The POZ compound wasmade by initiation of polymerization using a moiety containing theactivating group. However, Jordan, Hoogenboom and others have shown thatinitiation of polymerization with different activating groups canrequire greatly differing reaction conditions requiring extensivestudies to determine optimal reaction conditions. Thus if one choosesthe initiator method to make activated POZ compounds, one must conductexploratory work to determine appropriate reaction conditions for eachnew compound. Also the above compound is difunctional since it isterminated with a hydroxyl group rather than an inert group such as analkyl group. Also, as discussed below, initiation of polymerization withan alkyl halide does not proceed by a living-cation mechanism and thushigh polydispersities are found.

The NHS-activated glutarate derivative has been made by Myamoto andZalipsky. This derivative was prepared from a monofunctional POZ polymerhaving an OH group as the terminal activating group (M-Z-OH). However,glutarate and succinate derivatives have a hydrolytically unstable esterlinkage connecting the target molecule to the POZ compound. For example,the NHS-activated glutarate derivative will react with a targetmolecule, illustrated here as a protein, as shown:

M-PMOZ-O₂C—CH₂CH₂CH₂—CO₂—NHS+PROTEIN—NH₂→

→M-PMOZ-O_(w)C—CH₂CH₂CH₂CH₂—CONH—PROTEIN

As a result of the hydrolytically unstable ester linkage, the targetmolecule-POZ conjugate produced will not be stable in a biologicalsystem under physiological conditions, such as a human or other mammal,but will hydrolyze to cleave the POZ from the protein:

M-PMOZ-O₂C—CH₂CH₂CH₂—CONH—PROTEIN→

→M-PMOZ-OH+HO₂C—CH₂CH₂CH₂—CONH-PROTEIN

Furthermore, in the scheme above, when the target molecule-POZ conjugateundergoes hydrolysis due to the cleavage of the unstable ester linkage,the resulting target molecule will contain a “tag” or “hapten” which canlead to immunogenicity of the target molecule.

Finally, the orthopyridyl disulfide (OPSS) derivative has been made (G.Hsiue, et al., Bioconjugate Chem., 2006, 17, 781-786). This derivativecould in theory be coupled to a protein thiol group to give a disulfidelinkage, although this was not done by Hsiue, but it is known thatdisulfides are unstable and subject to ready reduction in plasma.

An additional problem hindering use of known POZ polymers formodification of target molecules is that some POZ polymers do notpossess a single active functional group; i.e., they are not“monofunctional”. Monofunctionality is necessary if one seeks to avoidcrosslinking and or incorporation of multiple target molecules on thepolymer backbone. For example, Jordan and his colleagues have publishedwork showing copolymerization of 2-ethyl-2-oxazoline with oxazolinemonomers containing functional groups. These pendent functional groupsare capable in some instances of being coupled to peptides. However,this technique was not designed to provide monofunctional POZ polymersbut rather produces multifunctional compounds with pendent groups alongthe backbone. Having multiple functional groups present in the POZbackbone can be advantageous in some instances, but would lead tocrosslinking and aggregate formation when coupling with multi-functionaltarget molecules, such as, but not limited to, polypeptides andproteins. Also there are instances when one would desire to have asingle target molecule coupled to a polymer, and multi-functional POZpolymers will not permit this.

Some of the above functional POZ compounds have the potential to becoupled to target molecules such as proteins and small molecule drugs.However, as work with polyethylene glycol-modified therapeutics hasshown, it is frequently necessary for commercial development ofpolymer-modified drugs to utilize polymers with molecular weights (MWs)as high as 40,000 Da or higher and with molecular weight distributionsor polydispersities (PDs) of less than 1.1. There has been a great dealof work showing that MWs and PDs in the above range cannot be achievedfor POZ with conventional techniques. It is generally seen that as themolecular weight of growing POZ chains reaches approximately 5,000 Da,the polydispersity increases appreciably. Side reactions, includingchain transfer, begin to grow in importance. Use of unusually lowpolymerization temperatures combined with reaction times of severalweeks has been shown to give acceptable PDs, but such conditions are notpractical for commercial-scale preparations (J. S. Park and K. Kataoka,Macromolecules, 39, 6622 (2006)). Hoogenboom, Schubert and colleaguesindicate that low-PD POZ can be prepared by using microwave irradiation,but again commercial-scale polymerizations are not available with thistechnique (R. M. Paulus, T. Erdmenger, C. R. Becer, R. Hoogenboom and U.S. Schubert, Macromol. Rapid Comm., 28, 484-491 (2007)). As aconsequence of the generally found broad polydispersities, thefunctional POZ compounds described to date are seriously limited for usein polymer therapeutics.

Yet another problem hindering use of POZ derivatives in modification oftarget molecules is the unavailability of a range of appropriateactivated POZ molecules capable of reaction with the target moleculesunder a range of conditions. Furthermore, the POZ molecules presentlyavailable are multifunctional or contain hydrolytically unstable bondswhen conjugated to target molecules, with the disadvantages associatedtherewith, or the active substituent is added during the initiationreaction, with the disadvantages associated therewith. Furthermore,pendent functionality has been described, but these derivatives aremultifunctional and not suitable for the current application.

SUMMARY OF THE INVENTION

The present disclosure provides monofunctional POZ derivatives having arange of active groups allowing conjugation of the monofunctional POZderivatives to a wide range of target molecules under a wide range ofreaction conditions to produce a hydrolytically stable targetmolecule-POZ conjugate; in certain embodiments, one target molecule isbound to the POZ derivative. The ability to provide monofunctional POZderivatives with a range of active groups capable of reacting with aselected group on a target molecule under different reaction conditionsprovides a significant advantage over the prior art since differenttarget molecules are sensitive to different reaction conditions and themost effective reaction conditions for conjugation of a POZ derivativeto a target molecule frequently vary depending on the nature of thetarget molecule and the group on the target molecule reacting with thePOZ derivative.

The present disclosure addresses the limitations of the previouslydescribed POZ polymers by providing a range of POZ polymers and POZderivatives not previously known in the art. Furthermore, the presentdisclosure provides monofunctional POZ derivatives having activefunctional groups on the terminal end thereof. Still further, thepresent disclosure addresses the limitations of the prior art byproviding synthesis methods for the disclosed POZ derivatives utilizingPOZ molecules with terminator end groups, such as, but not limited to,the hydroxyl group, that are readily available and can be obtained usingknown preparation chemistries. A wide variety of POZ derivatives canthen be prepared by coupling of small, active molecules to the availableterminal group in a step-wise fashion to generate the desired activefunctional groups on the POZ derivative (as described herein). Inaddition, the present disclosure addresses the limitations of the priorart by providing a hydrolytically stable target molecule-POZ conjugatethrough the use of the described monofunctional POZ derivatives. Such anapproach increases the in vivo half-life of the target molecule-POZconjugate and reduces the problems of immunogenicity related to “haptentagging” of the target molecule. The disclosed monofunctional POZderivatives, synthesis methods and resulting hydrolytically stabletarget molecule-POZ conjugates have not been appreciated in the art.

Therefore, the described monofunctional POZ derivatives and synthesismethods avoid the problems inherent in the art and provide a mechanismto produce hydrolytically stable target molecule-POZ conjugates in whichone target molecule is bound to the POZ derivative.

Any 2-substituted-2-oxazoline compound, such as but not limited to, PMOZand PEOZ, may be used to produce the POZ derivatives of the presentdisclosure, as discussed in more detail below. In certain embodiments,PEOZ or PMOZ are the 2-substituted-2-oxazolines POZ molecules. As isknown in the art, different alkyl groups in the 2-alkyl-2-oxazolinemolecules can provide differing solubilities, pharmacokinetics andmembrane permeating abilities to the POZ derivatives described herein.In addition, the nature of the repeating unit in the POZ polymerbackbone may be the same to produce a homopolymer (such as but notlimited to PMOZ and PEOZ) or at least one of the repeating units may bedifferent to provide for copolymers, such as, but not limited to, randomor block copolymers.

Furthermore, the present disclosure provides novel methods forsynthesizing POZ polymers with low polydispersity (PD) values anddecreased amounts of impurities produced by unwanted side reactions,such as, but not limited to, chain transfer. In one embodiment, thepresent disclosure describes novel methods for minimizing unwanted sidereactions, such as, but not limited to, chain transfer, allowing theproduction of POZ polymers of increased purity with low PD values. Inone embodiment, the methods of the present disclosure provide for POZderivatives with low PD values at increased MW values. In a furtherembodiment, POZ polymers are produced with decreased amounts ofimpurities. The novel methods provided for in the present disclosure arean improvement over the methods of the prior art and provide for largescale commercial preparation of POZ polymers suitable for use inmodification of a wide variety of target molecules.

Therefore, the present disclosure also provides POZ polymers ofincreased purity and with low PD values suitable for use inpharmaceutical applications. As is known in the art, PD values will varywith MW; in general, as the molecular weight increases the PD value alsoincreases. Using the methods of the present disclosure, POZ polymers ofvarious MWs can be produced on commercial scale with lower PD values ata given MW than can be produced using the commercially-applicablemethods of the prior art. For example, using the methods of the presentdisclosure, POZ derivatives of 20,000 Da MW or less can be produced withPD values of less than or equal to 1.1. In a further particularembodiment, the foregoing are produced with decreased amount ofimpurities. As is known in the art and illustrated in the Examplesherein, POZ derivatives synthesized using the methods of the prior artexhibit certain impurities that are seen as high MW shoulders and low MWtails in GPC traces. These impurities are generated, at least in part,through unwanted side reactions, such as, but not limited to, chaintransfer. As a result, the disclosed POZ derivatives are suitable foruse in modification of a wide variety of target molecules.

The present disclosure also provides for new POZ-target moleculeconjugates. The POZ conjugates of the present disclosure providebeneficial properties not present in prior polymer conjugates of theart. In one embodiment, the present disclosure provides methodologies ofattaching POZ and/or POZ derivatives to target molecules and theresulting POZ-target molecule conjugates. In one embodiment, thePOZ-target molecule conjugates are hydrolytically stable. Non-enzymaticand enzymatic methods for producing such conjugates are provided. In oneembodiment, the target molecules are biopharmaceuticals and lipids.Exemplary lipids include, but are not limited to, natural lipids, suchas, but not limited to, phospholipid, a glycerolipid, or a sterol lipid,as well as synthetic lipids. Exemplary target molecules include, but arenot limited to, erythropoietin, granulocyte colony stimulating factor,dynorphin A. lysozyme, human growth hormone, apomyoglobin and hyaluronicacid.

Through the use of the methods described herein, a range ofmonofunctional POZ derivatives with different active functional groupsare provided. Furthermore, the present disclosure provides synthesismethods to produce such monofunctional POZ derivatives in an efficientmanner. Finally, through the use of the monofunctional POZ derivativesdescribed, a hydrolytically stable target molecule-POZ conjugate may beproduced in which one target molecule is bound to the POZ derivative.

Methods of Synthesis of POZ Derivatives With Low PD Values

The current state of the art for polymerization of 2-aryl- and2-alkyl-2-oxazolines is derived from the publications of Kobayshi,Nuyken and Jordan (S. Kobayashi, E. Masuda, S. Shoda and Y. Shimano,Macromolecules, 1989, 22, 2878-2884; A. Gross, G. Maier and O. Nuyken,Macromol. Chem. Phys., 1996, 197, 2811-2826; and F. C. Gaertner, R.Luxenhofer, B. Blechert, R. Jordan and M. Essler, J. Controlled Release,2007, 119, 291-300). In these methods polymerization is initiated withan electrophile, such as an alkyl tosylate or alky triflate; in oneembodiment, methyl tosylate or methyl triflate is used. These strongelectrophiles are used to favor polymerization by a living-cationmechanism since this mechanism, in theory, gives no termination orchain-transfer reactions (Q. Liu, M. Konas and J. S. Riffle,Macromolecules, 1993, 26, 5572-5576) (see FIG. 1). However, it is knownfrom the prior art that chain transfer reactions do occur and that thereaction does not proceed strictly by the living cation mechanism. Ifweak electrophiles such as, but not limited to, alkyl halides are used,the reaction proceeds by a covalent mechanism with a consequentsignificant increase in PD. The prior art polymerization methods utilizechlorobenzene, dichlorobenzene or acetonitrile as solvent. Thepropagation phase is conducted at approximately 80° C. for approximately1-3 days. Termination is conducted by heating at 80-90° C. with aqueoussodium carbonate to give a hydroxyl terminal group or by reacting with asecondary amine such as morpholine or piperidine to give a terminaltertiary amine.

The use of these typical, prior art methods leads to the presence of ahigh-MW shoulder of approximately 5-10% and significant low-MW tailingin gel permeation chromatography. Such results have been noted in theart (see J. Park and K. Kataoka, Macromolecules, 2006, 39, 6622-6630.).It is generally stated in the literature that this broadening of the MWdistribution is due to chain transfer proceeding through anelimination-dimerization mechanism, although structural details andexperimental support for this process are limited (M. Litt, A. Levy andJ. Herz, J. Macromol. Sci.-Chem., 1975, A9, 703-727). To the extent thatchain transfer reactions do occur, such reactions cannot be consideredto be truly living polymerizations. Therefore, it would be beneficial toreduce the occurrence of unwanted side reactions such as chain transfer.

The applicants have clarified the details of theelimination-dimerization mechanism, provided experimental support forthe mechanism, and proposed implications of the mechanism regarding thetermination step. This latter advance is particularly important becauseit shows why certain termination reactions fail and it leads us tochoose termination reactions that succeed. Such a finding has not beendescribed in the art and it provides guidance in creating syntheticmethods that minimize the occurrence of unwanted side reactions and thatyield the desired terminated products.

As discussed herein, the use of the prior art methods produced a POZproduct that contained a high MW shoulder of approximately 5-10% of thetotal mass of the POZ product. This high MW shoulder contributes to theunacceptable PD values obtained using synthetic methods of the priorart. The high MW shoulder observed in the methods of the prior art iscomposed, at least in part, of a high-MW dimer that is formed during thepolymerization and/or termination steps (see FIG. 2). Theelimination-dimerization mechanism predicts that if chain transferoccurs during the termination step, the material in the high MW shoulderwould be approximately double the MW of the desired product.Furthermore, if chain transfer occurs during the propagation step, a newpolymer chain will be initiated, and since monomer concentration is lessat this point, the MW of this polymer will be less than that of the bulkof polymer. In addition, since this new polymer chain results from chaintransfer, it must be initiated by a proton, rather than by methyl, andthus the MALDI spectrum of this polymer will show a set of peaks 14 Daless than that of the main peak.

Methods for synthesizing POZ polymers and POZ derivatives with low PDand methods of using the foregoing, as well as benefits of using theforegoing, are described in U.S. application Ser. No. 12/529,001; thisapplication is hereby incorporated by reference for such teachings, suchas pages 15-20 and the examples recited therein.

Methods of Synthesis of Monofunctional POZ Derivatives

The present disclosure provides novel methods to synthesize thedescribed monofunctional POZ derivatives. The novel synthetic methodsare referred to herein generally as the building block method. In oneembodiment of the building block method, a one-step synthetic method isdisclosed. In an alternate embodiment of the building block method, atwo-step method is disclosed. In a further alternate embodiment of thebuilding block method, a living polymer synthesis is described. Each ofthe methods will be described in more detail below. In any of theforegoing embodiment, POZ polymers and POZ derivatives with low PDvalues may be used.

Building Block One-Step Method

In a first embodiment of the building block approach, a one-step methodis disclosed. In the one-step method, a range of monofunctional POZderivatives is generated in a single step through reaction between asingle terminally-functionalized POZ molecule and a set of compoundscontaining the desired active group. In this way a singleterminally-functionalized POZ molecule can be converted to a range ofactivated monofunctional POZ derivatives. This approach means that oneneed only optimize the polymerization chemistry for production of asingle monofunctional POZ derivative. The POZ terminal group (Y below)is chosen carefully to make possible this range of reactions to providea range of active groups. The one-step method can be represented asfollows in its general form in Scheme 1:

R₁-POZ-Y→R₁-POZ-P_(p)-Q_(q)-X  1

Scheme 1 Where

-   -   POZ is —[N(COR₇)CH₂CH₂]_(n)—;    -   R7 is independently selected for each repeating unit from an        unsubstituted or substituted alkyl, alkenyl or aralkyl group, in        one embodiment having from 1 to 12 carbons;    -   R₁ is hydrogen or unsubstituted or substituted alkyl, alkenyl or        aralkyl group groups;    -   R₂-R₄, R₁₁ and R₁₄-R₁₅ are each independently selected from        hydrogen or unsubstituted or substituted alkyl, alkenyl or        aralkyl group groups, in one embodiment having from 1 to 10        carbons;    -   R₈ is —C₆H₁₀—CH₂— (cyclohexylmethylene);    -   R₂₃ is unsubstituted or substituted alkyl, alkenyl, alkynyl or        aralkyl group groups, in one embodiment having from 1 to 10        carbon atoms, or substituted or unsubstituted aryl groups;    -   Y is —OH, —SH, —S—(CH₂)_(k)—CO₂H, piperazinyl, substituted        piperazinyl, substituted piperidinyl, or —NHR₂;    -   P is a linking group; P can be any group capable of forming the        linkages shown in scheme 1 and may be selected depending on the        chemistry of the groups with which it forms a linkage;        representative P groups include, but are not limited to, —O—,        —S—, —NH—, —NR₁₁— or unsubstituted heterocyclyl, such as, but        not limited to, piperazinyl (NC₄H₈N);    -   Q is a linking group; Q can be any group capable of forming the        linkages shown in scheme 1 and may be selected depending on the        chemistry of the groups with which it forms a linkage;        representative Q groups include, but are not limited to, an        unsubstituted or substituted alkyl, alkenyl, heterocyclyl or        aryl group, —(CH₂)_(m)-CONH-(CH₂)_(m)—,        —NH—(CH₂)_(m)-NHCO-(CH₂)_(m), —CO-(CH₂)_(m)—, —CO—C₆H₄—, or        —CO—R₈, -(R₁₅)_(m)— or —(CR₃R₄)_(m);    -   n is an integer from 3 to 1000;    -   k and m are integers independently selected from 1 to 10;    -   p and q are integers independently selected from zero or one;

X is an active functional group capable of forming a linkage with atarget molecule to produce a hydrolytically stable target molecule-POZconjugate.

In one embodiment, the active functional group is selected from thefollowing general classes of compounds: aldehydes (—CHO), activecarbonates (—O—CO—Z), maleimides, sulfonate esters (—OSO₂-R₂₃),including but not limited to tresylate (2,2,2-trifluoroethanesulfonate)and mesylate (—O—SO₂—CH₃ or —OMs), hydrazides (—CONHNH₂), epoxides,iodoacetamides, alkynes, azides (—N₃), isocyanates (—OCN), cyanates(—NCO), isothiocyanates (—SCN), thiocyanates (—NCS), nitriles (—CN),carbonyldiimidazole derivatives, vinylsulfones, carboxylic acid halides,active esters (—CO—Z) and carboxylic acids (—CO₂—H); and

Z is an activating group of which there are many known in the artincluding N-succinimidyloxy, chlorine, bromine, sulfo-N-succinimidyloxy,p-nitrophenoxy, 1-imidazolyl, and 1-benzotriazolyloxy;

The active functional group may also be protected to yield a protectedactive functional group by methods known in the art. For example, anacetal [CH(OR₁₄)₂] is an exemplary protecting group, which can behydrolyzed to produce an aldehyde group. The active functional group maybe substituted with groups, such as but limited to, those groupsdescribed with respect to a substituted alkyl group and substituted andunsubstituted alkyl, alkenyl, alkynyl, aralkyl or heterocycloalkylgroups. Furthermore, the active functional group includes thosecompounds that may be converted to an active functional group. Forexample, the X group may include a compound that is modified by alinkage that is susceptible to hydrolysis under certain reactionconditions (such as those used to join the POZ derivative to a targetmolecule), thereby cleaving the linkage and exposing the activefunctional group to react with a group on the target molecule.

In Scheme 1, the reactant R₁-POZ-Y is the direct product ofpolymerization of POZ and the Y group is capable of being converteddirectly to a series of monofunctional POZ derivatives capable offorming a hydrolytically stable target molecule-POZ conjugate. Forexample, in one embodiment Y equals —OH and is obtained when the POZpolymerization reaction is terminated with hydroxide. In an alternateembodiment, Y equals —NHR₂ and is obtained when the POZ polymerizationreaction is terminated with a compound containing an amino group, R₂NH₂.Other useful amine terminating agents providing useful Y groups arepiperazine or a substituted piperazine such as 1-piperazinepropanol(H—NC₄H₈N—CH₂CH₂CH₂—OH) Substituted piperidines are also useful sincethese provide the rapid termination usual for amines, and they alsointroduce a range of functional groups. Commercially availablesubstituted piperidines include 4-piperidine butyric acid, 3-piperidinecarboxylic acid and 4-piperidine methanol. In an alternate embodiment, Yequals —S—CH₂CH₂—CO₂H and is obtained with the POZ polymerizationreaction is terminated with ⁻S—CH₂CH₂—CO₂—CH₃ (followed by hydrolysiswithout isolation).

Various exemplary reactions illustrating the preparation via theone-step method of R₁-POZ-X derivatives are illustrated below. In thereactions presented below, R₁ and R₇ are methyl and Y is —OH or —NHR₂,where R₂ is CH₃.

The structures above can be seen to fall within the description ofScheme 1. For example, for the active carbonate above (M-PMOZ-OCO₂—NHS)R₁ and R₇ are methyl, p and q are zero, and X is the succinimidylcarbonate. For the active ester above (M-PMOZ-O—CH₂—CO₂NHS) R₁ and R₇are methyl, P is —O—, p is 1, Q is —CH₂—, q is 1, and X is —CO₂NHS.

The monofunctional POZ derivatives with the active groups describedabove provide a number of useful and differing properties, allowing forthe selection of a particular monofunctional POZ derivative with adesired active functional group based on the nature of the targetmolecule and the desired reaction conditions. For example, when theactive functional group is an aldehyde, the monofunctional POZderivative reacts predominately with the N-terminal amine of the targetmolecule in a defined pH range to form an imine (which is typicallyreduced with borohydride to a secondary amine). When the activefunctional group is an active ester, the monofunctional POZ derivativereacts predominately with amines, including, but not limited to,non-terminal lysine groups on the target molecule. Likewise, when theactive functional group is an active carbonate or tresylate, themonofunctional POZ derivative reacts readily with amines, but withreaction conditions and selectivity different from active esters andaldehyde. Furthermore, when the active functional group is avinylsulfone or maleimide the monofunctional POZ derivative reactspredominately with thiols, but the reaction conditions differ for eachof these groups, providing a range of reaction conditions appropriatefor a range of target molecules.

Importantly, each of the monofunctional POZ derivatives formed using thesynthetic scheme above is capable of forming a hydrolytically stabletarget molecule-POZ conjugate.

Building Block Two-Step Method

In an alternative embodiment, a two-step synthesis method is disclosed.In the first step of the two-step method, an initial polymer product(R₁-POZ-Y, below), prepared by polymerization as described above, isreacted with a desired compound to produce a POZ intermediate(R₁-POZ-Y′, below). In the second step of the two-step method, this POZintermediate is further reacted with a range of compounds comprising arange of active functional groups to form a series of monofunctional POZderivatives (R₁-POZ-X, below) capable of forming hydrolytically stabletarget molecule-POZ conjugates. The two-step synthetic method offers theadvantage of providing a range of monofunctional POZ derivatives usingonly two reactions and starting from a single initial polymer product(R₁-POZ-Y), thus minimizing the need to optimize polymerizationconditions for multiple polymer products. The two-step method providesmonofunctional POZ derivatives not available by the one-step method. Inits most general form, the transformations of the building blocktwo-step method are illustrated in Scheme 2 below.

P₁POZ-Y→R₁-POZ-Y′→R₁-POZ-X  II

Scheme 2

The building block two-step method can be presented in a detailed formas follows in Scheme 3. In Scheme 3, Y′ is an active group, including,but not limited to, active esters and active carbonates, capable ofreacting with a functional nucleophile, represented by the T group.

Step 1

R₁-POZ-Y→R₁-POZ-P_(p)-Q_(q)-Y′

Step 2

R₁-POZ-P_(p)-Q_(q)-Y′+T-U_(u)-X→R₁-POZ-P_(p)-Q_(q)-W_(w)-U_(u)-X  III

Scheme 3 Where

-   -   R₁-R₄, R₇, R₈, R₁₁, R₁₄-R₁₅, R₂₃, POZ, P, Q, k, m, n, p, q, Y,        and X are as described above;    -   U is a linking group; U can be any group capable of forming the        linkages shown in scheme 3 and may be selected depending on the        chemistry of the groups with which it forms a linkage;        representative U groups include, but are not limited to,        including, but not limited to —(R₁₆)_(o)—, —(CR₅R₆)_(o)—,        —NH—R₂₁—NHCO—R₂₂—;    -   o is an integer from one to ten;    -   w and u are integers independently selected from one or zero;    -   R₅, R₆, R₁₆, R₂₁ and R₂₂ are each independently selected from        hydrogen or unsubstituted or substituted alkyl, alkenyl or        aralkyl groups, in one embodiment having from 1 to 10 carbon        atoms;    -   R₁₇ is selected from hydrogen or unsubstituted or substituted        alkyl, alkenyl or aralkyl group groups, in one embodiment having        from 1 to 10 carbon atoms, or substituted or unsubstituted aryl        groups.

Y′ and T are a reactive pair that react to form in a linkage W which ishydrolytically stable, wherein the Y′ and T reactive pair and theresulting W linkage are selected from those groups and linkages shown inTable 1. Y′ and T groups may be reversed without affecting the nature ofthe W linkage.

TABLE 1 Some possible T-Y′ pairs and resulting W linkages T group Y′Group W Linkage —NH₂ Any active carbonate Urethane (such as, but not(—NH—CO—O—) limited to, —O—CO—O—Z) —OH isocyanate (—NCO) Urethane(—NH—CO—O—) —NH₂ any active ester or Amide (—NH—CO—) acid halide (suchas, but not limited to, —CO—O—Z, —CO—Cl and —CO—Br) —NH₂ NCO Urea(—NH—CO—NH—) —NCS —NH₂ Thiourea (—NH—CS—NH—) halides —Cl —OH Ether (—O—)or —Br —OH —OSO₂—R₁₇ Ether (—O—) halides —SH Thioether (—S—) O—SO₂—R₁₇—SH Thioether (—S—) halides —NH₂ Amine (—NH—) O—SO₂—R₁₇ —NH₂ Amine(—NH—) —SH —NCO —S—CO—NH— —SH —OSO₂—R₁₇ Thioether (—S—)

An exemplary reaction illustrating the preparation of a hydrolyticallystable R₁-POZ-P_(p)-Q_(q)-W_(w)-U_(u)-X derivative is illustrated below.In this reaction R₁ is hydrogen and R₇ is methyl, Y is —OH, p and q arezero, U is —CH₂CH₂—, u is one, the reactive pair T and Y′ are —NH₂ and—O—CO₂—NHS, respectively, which form the urethane W linkage, w is one,and X is an acetal (protected aldehyde):

H-PMOZ-OH→M-PMOZ-O—CO₂-NHS

H-PMOZ-O-CO₂-NHS+NH₂-CH₂CH₂—CH(OEt)₂→

H-PMOZ-OCONH-CH₂CH₂-CH(OEt)₂,

which after hydrolysis, yields

H-PMOZ-OCONH-CH₂CH₂-CHO.

Another example of Scheme 3 is illustrated below. In this reaction R₁and R₇ are methyl, Y is —OH, P is —O—, p is one, Q is —CH₂CH₂—, q isone, the reactive pair T and Y′ are —NH₂ and —CO₂H, respectively, whichform the amide (—CONH) W linkage, w is one, U is —CH₂CH₂—, u is one, andX is acetal (protected aldehyde):

M-PMOZ-OH→M-PMOZ-O-CH₂CH₂-CO₂H

M-PMOZ-O-CH₂CH₂-CO₂H+NH₂-CH₂CH₂-CH(OEt)₂→

M-PMOZ-O-CH₂CH₂-CONH-CH₂CH₂-CH(OEt)₂

which after hydrolysis, yields

M-PMOZ-O-CH₂CH₂-CONH-CH₂CH₂-CHO

As discussed above, each of the active functional groups of Scheme 3 hasunique advantages and specificities in reacting with target molecules.Furthermore, the reactivity of the T and Y′ groups towards one anothermay be controlled through the nature of the Q and U groups. Byincreasing the chemical distance between the T and Y′ and/or the T and Xgroups by increasing the size of the Q and U groups as described, thereactivity of the Y′ and T groups is altered. Furthermore, thereactivity of the active functional group X towards the target moleculemay be similarly modulated.

As with the one-step method, each of the monofunctional POZ derivativesformed using the two-step synthetic scheme above is capable of forminghydrolytically stable target molecule-POZ conjugates.

The Living-Polymer Method

In a further alternative embodiment of the invention, small, reactivemolecules may be used to terminate oxazoline polymerization to directlyprovide monofunctional POZ derivatives, which can react with targetmolecules to form a hydrolytically stable target molecule-POZ conjugate.This method is referred to as the living polymer method. The livingpolymer method can be presented in its most general terms as follows inScheme 4:

R₁-POZ⁺+Nuc-Q_(q)-X→R₁-POZ-C-Q_(q)-X  IV

Scheme 4 Where

R₁-R₄, R₇, R₈ and R₁₄-R₁₅, R₂₃, POZ, Q, k, m, n, q, Y, and X are asdescribed above;

POZ⁺ represents the cation —[N(COR₇)CH₂CH₂]_(n) ⁺ formed duringoxazoline polymerization;

R₁₉ is selected from hydrogen or unsubstituted or substituted alkyl,alkenyl or aralkyl group groups, in one embodiment having from 1 to 10carbon atoms; and

Nuc is a nucleophile capable of terminating the living POZpolymerization reaction by interacting with the terminal cation—N(COR₇)CH₂CH₂ ⁺ to form the hydrolytically stable linkage C, whereinthe Nuc group and the resulting C linkage can be selected from thosegroups and linkages shown in Table 2.

TABLE 2 Some possible Nuc groups and C linkages Nuc Group C Linkage—NHR₁₉ Amine (—NR₁₉—) —SH Thioether (—S—) —NH₂ Amine (—NH—) piperazinePart of ring structure piperidine Part of ring structure

An exemplary reaction illustrating the preparation of theR₁-POZ-C-Q_(q)-X derivative is given below. In the reaction presentedbelow, R₁ and R₇ are methyl, Nuc is —NH₂, C is —NH—, Q is —CH₂—, q isone, and X is —CO₂H (note that the methyl ester is hydrolyzed during thereaction below):

CH₃-PMOZ⁺+^(−S—CH) ₂—CO₂—CH₃→CH₃-PMOZ-S-CH₂-CO₂H

Contrary to the suggestions in the art, the present disclosure showsthat cation trapping with alkoxides, such as —O—CH₂—CO₂—CH₃, does notresult in the desired product.

As stated in Table 2, the C linkage is incorporated into the piperazineand piperidine ring structure when these compounds are used as the Nucgroup. Examples of the C linkage and the structures resulting from usingpiperazine and piperidine as the Nuc group are provided below.

In one embodiment of this reaction, a mercaptide compound is used toterminate the oxazoline polymerization. In this method, oxazolinepolymerization is initiated as described herein to form a POZ polymerwith an oxazolinium cation at the terminating end of the POZ polymer.The reaction is terminated by adding a nucleophilic mercaptide moleculeto the reaction, thereby terminating the living POZ polymerization. Themercaptides molecule comprises an active functional group (the activefunctional group may be protected as described herein) capable ofreaction with a group on a target molecule to form a hydrolyticallystable linkage.

In a specific embodiment of this method, the mercaptides has thestructure R₂₅S-D_(d)-X, wherein:

-   -   X is as defined above;    -   R₂₅ is a metal; in one embodiment, R₂₅ is Li, Na or K;    -   D is a linking group, including but not limited to, an        unsubstituted or substituted alkyl, alkenyl, heterocyclyl or        aryl group, —(CH₂)_(b)—CONH—(CH₂)_(b)—,        —NH—(CH₂)_(b)—NHCO—(CH₂)_(b),—CO—(CH₂)_(b)—, —CO—C₆H₄—, or        —CO—R₂₆, or —(CR₂₇R₂₈)_(b);    -   R₂₇ and R₂₈ are each independently selected from hydrogen or        unsubstituted or substituted alkyl, alkenyl or aralkyl group        groups, in one embodiment having from 1 to 10 carbons;    -   R₂₆ is —C₆H₁₀—CH₂— (cyclohexylmethylene);    -   d is 0 or 1; and    -   b is an integer from 1 to 10.

In one embodiment, the active functional group is a protected functionalgroup or a compound that may be converted to an active functional group.As discussed above, each of the active functional groups has uniqueadvantages and specificities in reacting with target molecules. Asabove, each of the monofunctional POZ derivatives formed using thesynthetic scheme above is capable of forming a hydrolytically stabletarget molecule-POZ conjugate.

Specific POZ Derivatives

The present disclosure describes a variety of monofunctional POZderivatives which can be prepared by the methods described above.Furthermore, the present disclosure describes a number of compoundsuseful in the synthesis of the monofunctional POZ derivatives of thepresent disclosure.

In one embodiment, the monofunctional POZ derivatives are described bythe general formula (I), (III) or (IV):

R₁-POZ-P_(p)-Q_(q)-X  (I)

R₁-POZ-P_(p)-Q_(q)-W_(w)-U_(u)-X  (III)

R₁-POZ-C-Q_(q)-X  (IV)

Wherein the definitions in the general formulas (I), (II) and (III) areas provided for above with reference to Schemes 1-4.

In addition, a number of specific structures for the monofunctional POZderivatives of the present disclosure are provided below. Thesestructures are listed for exemplary purposes only and are not meant tolimit the scope of the monofunctional POZ derivatives described herein.As above, when referred to below, the definitions provided in Schemes1-4 above are applicable to the structures below; the definitions belowalso apply where applicable. In addition, for all the structuresprovided below, the R₁ group is understood to be included at theposition of the initiator group (to the left of the POZ group).

-   -   R₉ is a linking moiety such as —(R₁₆)_(O)— or —NH—R₂₁—NHCO—R₂₂—;    -   G is an unsubstituted or substituted aryl group or a substituted        or unsubstituted alkyl, alkenyl or alkynyl group, such as, but        not limited to, a fluoroalkyl group; and    -   Ar is an unsubstituted aryl or substituted aryl group.

In one embodiment, the present disclosure provides for monofunctionalPOZ derivatives made by the building block one-step method. Exemplarystructures derived by this route include, but are not limited to, thefollowing structures.

-   -   POZ-P—(CR₃R₄)_(m)—CH(OR₁₄)₂    -   POZ-P—(CR₃R₄)_(m)—CHO and POZ-NHCO—C₆H₄—CHO    -   POZ-P—(CR₃R₄)_(m)—CO₂H    -   POZ-P—(CR₃R₄)_(m)——CO—Z    -   POZ-P—(CR₃R₄)_(m)—CO—NH—NH₂    -   POZ-O₂C—O—Z    -   POZ-O—SO₂-G

-   -   POZ-P—CH₂CH₂—SO₂CH═CH₂ and POZ-NHCO—C₆H₄—SO₂—CH═CH₂    -   POZ-NH—CO—C₆H₄—NHCO—CH₂—I    -   POZ-P—(CR₃R₄)_(n)—CCH

In another embodiment, the present disclosure provides formonofunctional POZ derivatives made by the building block two-stepmethod utilizing nucleophilic displacement on a POZ sulfonate ester (anintermediate derived from POZ-OH):

-   -   POZ-N₃    -   POZ-P—(CR₃R₄)_(n)—CH(OR₁₄)₂    -   POZ-P—(CR₃R₄)_(m)—CHO    -   POZ-OCN    -   POZ-SCN    -   POZ-CN    -   POZ-P—(CR₃R₄)n—CCH    -   POZ-P—(CR₃R₄)_(n)—CO₂H and POZ-P—(CR₃R₄)_(n)—CO—Z    -   POZ-P—Ar—CO₂H and POZ-P—Ar—CO—Z

In another embodiment, the present disclosure provides formonofunctional POZ derivatives made by the building block two-stepmethod utilizing nucleophilic displacement on a POZ active carbonate (anintermediate derived from POZ-OH):

-   -   POZ-OCONH—(CR₃R₄)_(n)—CO₂H    -   POZ-OCONH—(CR₃R₄)_(n)—CO—Z    -   POZ-OCONH—C₆H₄—CHO

In another embodiment, the present disclosure provides formonofunctional POZ derivatives incorporating maleimides made bynucleophilic substitution on any of the above active esters:

Where

-   -   POZ-L—CO— is derived from any of the herein described active        carboxylate esters; and    -   L is any of the linking moieties shown above that link POZ to        the carboxyl group and includes —P—(CR₃R₃)_(m)—, —P—Ar—,and        pyridinium —NC₅H₄ ⁺—.

These maleimides can be seen to fit the abovePOZ-P_(p)—Q_(q)—W_(w)—U_(u)—X formula in which L comprises theP_(p)—Q_(q) segment, —CONH— comprises the W_(w) segment, and R₉comprises the U_(u) segment.

In another embodiment, the present disclosure provides for POZderivatives made by the living cation method utilizing nucleophilicattack on the POZ cation generated during polymerization of2-alkyl-2-oxazoline:

-   -   POZ-P—(CR₃R₄)_(n)—CO₂H    -   POZ-P—Ar—CO₂H and POZ-P—Ar—CO—Z

All of the above POZ derivatives react with a group on a target moleculeto form a hydrolytically stable linkage between the target molecule andthe PO derivative.

Specific POZ Derivatives from Piperidines or Piperazines

As discussed herein, the living POZ cation can be terminated with asubstituted or unsubstituted piperidine or piperazine or derivatives ofthe foregoing. The substitutions include, but are not limited to, thosegroups described with respect to a substituted alkyl and substituted andunsubstituted alkyl, alkenyl, aralkyl or heterocycloalkyl. These POZderivatives are difficult to illustrate with the above Schemes 1-4because the linking group, designated C in Scheme 4, is part of thepiperidine or piperazine ring structure. For example, the POZ cation canbe trapped with 4-piperidine methanol to yield a POZ alcohol, with4-piperidine butyric acid to yield a POZ carboxylic acid, or withpiperazine itself to yield a POZ amine:

Terminations with such piperidines and piperazines are useful becausethe strong nitrogen nucleophile gives rapid and clean termination tointroduce a terminal active functional group. At least four piperidineand piperazine derivatives are commercially available, including1-piperazinepropanol, 4-piperidine butyric acid, 3-piperidine carboxylicacid and 4-piperidine methanol, and others could be readily synthesized.

It is to be understood that any of the POZ derivatives described abovewhich are prepared from POZ alcohols, acids or amines could be preparedfrom such piperidines or piperazines in which the nitrogen-containingring provides the alcohol, acid or amine. For example, the followingcompounds can be prepared from the above POZ terminated with4-piperidine methanol:

These compounds in turn could be converted to a range of usefulderivatives including acetals, maleimides, and active esters:

Use of the POZ Derivatives

The described monofunctional POZ derivatives, through the activefunctional groups, may be used to produce a hydrolytically stable targetmolecule-POZ conjugate. In addition, using the monofunctional POZderivatives described herein, in certain embodiments one target moleculeis bound by each POZ derivative. The variety of active functional groupspresent in the monofunctional POZ derivatives allow for themonofunctional POZ derivatives to be coupled to a variety of groups onthe target molecule using a variety of reaction chemistries. Forexample, when the active functional group is an aldehyde, themonofunctional POZ derivative reacts predominately with N-terminalamines of a target protein molecule in a defined pH range. When theactive functional group is an active ester, the monofunctional POZderivative reacts predominately with amines, including, but not limitedto, lysine groups on a target molecule. Likewise, when the activefunctional group is an active carbonate or Cresylate, the monofunctionalPOZ derivatives react with amines, but with reaction conditions andselectivity different from active esters and aldehyde. Furthermore, whenthe active functional group is a vinylsulfone, maleimide oriodoacetamide, the monofunctional POZ derivative reacts predominatelywith thiols, but the reaction conditions differ for each of thesegroups, providing a range of reaction conditions appropriate for a rangeof target molecules.

Importantly, each of the monofunctional POZ derivatives formed using thesynthetic scheme above is capable of forming hydrolytically stabletarget molecule-POZ conjugates.

Physiologically active target polypeptide and protein target molecules,also referred to herein as biopharmaceuticals, have played importantroles in clinical therapeutics. Some examples of biopharmaceuticals thatact in-vivo are the interleukins, granulocyte colony stimulating factor(GCSF), granulocyte/macrophage colony stimulating factor (GMCSF),macrophage colony stimulating factor (MCSF), erythropoietin (EPO), α-,β- and γ-interferons, insulin, growth hormones, somatostatin,vasopressin, modified peptides, animal and microorganism derivedpeptides and human and animal antibodies and antibody fragments.

In one embodiment, the target molecule is a polypeptide. For example, amonofunctional POZ derivative may be coupled to the therapeuticallyimportant protein, such as, but not limited to, GCSF or EPO. Oneexemplary reaction for forming a POZ-GCSF conjugate is illustratedschematically below by the following reaction:

M-POZ-OCO—O—NHS+GCSF—NH₂→

M-POZ-OCONH—GCSF

In this embodiment, the amine shown on GCSF represents one of theseveral available lysine groups.

Similarly GCSF contains an available thiol group, and under the properconditions a monofunctional POZ derivative with maleimide as the activefunctional group can react with this thiol group.

POZ derivatives can be coupled to peptides as well. For example, amonofunctional POZ derivative with an active ester as the activefunctional group can couple to the available amino groups on insulin:

M-POZ-O—CH₂—NHS+Insulin—NH₂→

M-POZ-O—CH₂—CONH—Insulin

In an alternate embodiment, the target molecule is a small molecule,drug or a diagnostic agent.

The foregoing examples show POZ-target molecule conjugates between themonofunctional POZ derivatives described herein and in U.S. patentapplication Ser. No. 12/529,001. However, other POZ derivatives may beused in the POZ-target molecule conjugates described herein. In analternate embodiment the POZ component used in the formation of thePOZ-EPO conjugate is a POZ derivative as described in InternationalPatent Application Nos. PCT/US2008/078159, PCT/US2009/030762 orPCT/US2009/050286. Furthermore, the active functional group may be atthe initiator end, the terminal end or at a pendent position.Furthermore, the POZ portion of the POZ derivative may be a homopolymeror a random or block copolymer as described in International PatentApplication Nos. PCT/US2008/078159, PCT/US2009/030762 orPCT/US2009/050286. The foregoing patent applications are herebyincorporated by reference for such teachings.

Polymer-modified biopharmaceuticals have proven to have great utility inmodern clinical therapeutics. These preparations have been shown to havegreat versatility in the treatment of blood disorders such as anemia andneutropenia, viral infections, growth disorders and autoimmunedisorders. However, despite this promise the use of biopharmaceuticalshas been noted to have several shortcomings.

First, the bioactivity of many biopharmaceuticals is limited by ashort-half life in blood. For example, in order to reach clinicallyoptimal levels, EPO must be injected 2-3 times a week to anemia patientsor reticulocyte levels in the blood will fall rapidly. Frequentadministration to achieve maximum efficacy is problematic because ofpoor patient compliance. Bioactivity is limited by rapid metabolism,decomposition by proteolysis and binding to serum albumin. Physicalchanges (aggregation or denaturation) and chemical changes (oxidation ormodification of chemical bonds in general) of the protein molecule canoccur under unfavorable storage and exposure conditions. At elevatedtemperatures (above 8° C.) unfolding of the protein occurs and thisleads to a loss in biological activity. Denaturation may lead toaggregation and particulate formation.

Second, side effects have been associated with frequent administrationof many biopharmaceuticals. For EPO, such side effects include, but arenot limited to, inflammation and infection at the site ofadministration. Such side effects cause additional health problems forthe patient and further contribute to poor patient compliance.

Third, antigenicity has been observed as a recurring side effectassociated with a number of natural or recombinant biopharmaceuticals.Antibodies are produced in-vivo and can lead to neutralization of thebiopharmaceutical and in some cases also create allergic reactions. Inthe former instance, higher doses and/or more frequent administration ofbiopharmaceuticals may circumvent the neutralizing effect of antibodies.However, higher doses may aggravate the observed side effects andresults in increases in dosing frequency may result in reduction ofpatient compliance.

To solve the above mentioned problems, a number of strategies have beenemployed. Entrapment of the biopharmaceutical in an inertphosphotidylcholine based liposome, vesicle or nanocapsule has beenused. The inert carrier will accumulate in the reticuloendothelialsystem (RES).

In addition, bonding of the biopharmaceutical to a macromolecule orpolymer substrate such as albumin, polylysine, hyaluronic acid orpolyethylene glycol (PEG) has been used.

Covalent modification of proteins increases the protein's effective sizeand reduces renal clearance (Abuchowski et al., 1984; Hershfield, 1987;Meyers et al., 1991). Polymer conjugation also improves in-vivostability (protection from proteases), improves protein solubility andlowers antigenicity (Katre et al., 1987; Katre, 1990).

Traditionally these modifications have been made by reacting terminalelectrophiles on the polymer with nucleophiles on the protein(non-enzymatically mediated reactions). Conjugation occurs at thea-amino group on the N-terminal amino acid or on the s-amino groups oflysine within the polypeptide chain. These reactions typically givenon-specific attachment of the macromolecule or polymer substrate to thebiopharmaceutical and may result in significant loss in bioactivity ofthe biopharmaceutical. In addition, bonding of the biopharmaceutical toa macromolecule or polymer substrate can be accomplished usingenzymatically mediated reactions. The latter approach provides thebenefit of increased specificity in the conjugation reactions, which maylead in increased activity in vivo.

Because of the success of polymer-modified therapeutics andbiopharmaceuticals in general, for example EPO, it is of interest toexpand the range of polymers suitable for such applications, especiallyto provide polymers having properties not possessed by polymers of theprior art. The present disclosure provides methodologies of attachingPOZ and/or POZ derivatives, to biopharmaceuticals and the resultingPOZ-biopharmaceutical conjugates. In one embodiment, thePOZ-biopharmaceutical conjugates are hydrolytically stable. Oneadvantage of POZ is that the hydrophilicity of the polymer can be variedby changing the nature of the R₇ group, such as, but not limited to,changing an alkyl group present in the R₇ position from methyl to ethylto propyl; these changes can lead to differences in the PK profile andto greater activity as shown by an increase in the area under theactivity curve.

POZ-Erythropoietin Conjugates

The present disclosure also provides for POZ-EPO conjugates.Erythropoietin (EPO) is a naturally formed glycoprotein that is producedin the kidney and which functions as a colony stimulating factorinvolved with the regulation of red blood cells. Human EPO was firstcloned and amino acid sequence reported by Lin et al. (Proc Natl AcadSci USA 1985 82: 7582-4) and Jacobs K et al. (Nature 313: 806-810 1985)(each of which is incorporated herein by reference for such teaching).Human EPO is a four helix bundle, typical of members of thehematopoietic growth factor family. In contrast to the invariant aminoacid sequence, the carbohydrate structures are variable, a featurereferred to as micro-heterogeneity. The differences in carbohydratemoieties, in terms of the branching pattern, complexity, size and chargehas profound effects on the pharmacokinetics and pharmacodynamics ofEPO. The effects of different glycosylation patterns have been wellstudied (Darling et al 2002 Biochemistry 41: 14524-14531; Stoning et al1998 Br J Haematol 100: 79-89; Halstenson et al 1991 Clin Pharmacol Ther50: 702-712; Takeuchi et al 1990 J Biol Chem 265: 12127-12130) (each ofwhich is incorporated herein by reference for such teaching).

EPO acts on precursor cells in the bone marrow to produce mature redblood cells. rh-EPO has been manufactured using recombinant DNAtechnology through cloning of the EPO gene and expression in ChineseHamster Ovary (CHO) cells (Lin, U.S. Pat. No. 5,618,698). EPO isavailable in formulations such as EPOGEN® and PROCRIT® in 1 mL singledose or 2 mL multi-dose injection vials. These products have beensuccessfully used to treat anemia associated with myelosuppressionfollowing chemotherapy in cancer patients, chronic renal failure, and insome cases of HIV infected patients on antiviral therapy. EPO has beenadministered by intravenous, intramuscular and subcutaneous injection.The measurement of reticulocytes in total erythrocytes is an indicatorof EPO activity.

The current formulations of EPO have been associated with certainshortcomings, including those discussed above (for example, shorthalf-life, side effect associated with frequent administration andantigenicity).

Several patents have issued on coupling of PEG to EPO (U.S. Pat. Nos.6,340,742, 6,583,272, 7,074,755, 7,128,913 and applicationsWO/2002/049673, 2005/0170457, 2004/0082765, 2004/0266690 and2006/0276634).

The present disclosure provides methodologies of attaching POZ and/orPOZ derivatives, to EPO and the resulting POZ-EPO conjugates. In oneembodiment, the POZ-EPO conjugates are hydrolytically stable. In oneembodiment, the products obtainable according to the present disclosurecomprise polyoxazoline derivatives conjugated to EPO. Such POZ-EPOconjugates may be obtained by non-enzymatic reactions or enzymaticmediated reactions as described herein. The conjugates according to thepresent disclosure have increased residence time in blood, and produceless immunogenic responses compared to the corresponding unconjugatedEPO. In addition, the conjugates of the present disclosure havedifferent PK profiles as compared to unconjugated EPO.

Polyoxazolines have side chains that may sterically hinder theattachment of this polymer to buried amino acid residues on largeproteins, such as EPO. One would anticipate that the yields of such aconjugation would be low. Furthermore, it is also surprising that theconjugates obtained using sterically hindered POZ polymers and POZderivatives maintain their biological activities.

Furthermore, the present disclosure provides methodologies that can beapplied to the attachment of the POZ derivative to different amino acidresidues of EPO (whether natural or synthetic) and to that of EPOvariants and mimetics. For example, POZ polymers could be coupled to theN-terminus of EPO (Alanine 1) or lysine 52 of EPO. The amino acidsequence human recombinant EPO is provided in FIG. 3 (SEQ ID NO. 1).

In one embodiment, the POZ component used in the formation of thePOZ-EPO conjugate is a POZ derivative as described herein. In analternate embodiment the POZ component used in the formation of thePOZ-EPO conjugate is a POZ derivative as described in InternationalPatent Application Nos. PCT/US2008/078159 PCT/US2009/030762 orPCT/US2009/050286.

EPO used to produce the POZ-EPO conjugates may be either natural,recombinant or synthetically produced EPO; furthermore, polypeptideshaving EPO activity may also be used, including, but not limited to EPOpolypeptide differing by one or more amino acids from human EPO.Furthermore, EPO having different patterns of glycosylation may be used.

The following EPO polypeptides have the same amino acid sequence asrecombinant human EPO, but variations in the methods of productionprovide differing glycosylation patterns. Epoetin alfa and epoetin betaare described in U.S. Pat. Nos. 4,703,008 and 5,955,422. Epoetin alfaand epoetin beta are produced in chinese hamster ovary (CHO) cells.Epoetin alfa is available under the trade names procrit (Ortho Biotech),eprex (Johnson & Johnson), epogin (Chugai) or epogen (Amgen). Epoetinbeta is available under the trade name neorecormon or recormon(Hoffmann-La Roche). Epoetin omega described in U.S. Pat. No. 5,688,679is produced in baby hamster kidney cells (BHK-21). Epoetin omega isavailable under the trade names epomax (Elanex). Darbepoetin alfa(Amgen) is available under the trade name aransep (Macdougall I C,Kidney Int Suppl. 2002 May;(80):55-61). Darbepoetin alfa was designed tocontain five N-linked carbohydrate chains (two more than rhEPO). Theamino acid sequence differs from that of rhEPO at five substitutions(Ala30Asn, His32Thr, Pro87Val, Trp88Asn, Pro90Thr), thus allowing foradditional oligosaccharide attachment at asparagine residues at position30 and 88. Each of the references in this paragraph are herebyincorporated by reference for the teaching of the EPO amino acidsequence and methods of production.

In addition, forms of EPO with various point mutations have beendescribed (Elliot et al 1997 Blood 89: 493-502; Elliot et al 1996 Blood87: 2702-2713; Syed et al 1998 Nature 395: 511-516; O'Narhi et al 2001Protein Engineering 14: 135-140; Bill et al 1995 Biochimica etBiophysica Acta 1261: 35-43; Yamaguchi et al 1991 J Biol Chem 266:20434-20439; and US Patent Publication No. 20080194475). Such EPOvariants have been reported to have similar properties to native orrecombinant EPO. Each of the references in this paragraph are herebyincorporated by reference for the teaching of the EPO amino acidsequence and methods of production.

TGase Catalyzed Conjugate Formation

As discussed above, enzymatically mediated reactions may providebenefits over non-enzymatically mediated reactions.

One example of an enzymatically mediated reaction is based on use oftransglutaminase (TGase). TGase, obtained from guinea pig liver orStreptomyces mobaraensis, is used in the conjugation process tofacilitate the attachment of a polymeric amine to the glutamine residueof a peptide or protein. In this reaction, the enzyme effectivelyreplaces the amine of the glutamine with the amine group from thepolymeric amine.

This TGase approach has been used to attach PEG, polylysine andpolysaccharides to proteins. The attachment of PEG amine tobiopharmaceuticals using TGase has been reported by Fontana et al.,Advanced Drug Delivery Reviews, 60 (2008) 13-28. U.S. Pat. No. 6,010,871(January 2000) broadly covers the bonding of polylysine, PEG andmonoclonal antibodies to proteins and peptides using TGase. U.S. Patentapplication 2006/0116322 (June 2006) is for a composition of EPOconjugated with polyalkylene oxide (PEG and propylene glycol) usingTGase. A more recent application WO 2008/017603 (February 2008) is for acomposition of G-CSF and PEG using TGase. The attachment ofpolysaccharides such as dextran, carboxymethylcellulose and polydextroseto bovine pancreatic trypsin through a transglutaminase-catalysedreaction has been reported by M. Villalonga et al. (World Journal ofMicrobiology and Biotechnology, 2006). The use of cationic saccharidessuch as polylysine-dextran in the conjugation on to β-lactoglobulin withthe aid of the TGAse enzyme was reported by T. Ikeuchi et al. (Biosci.Biotechnol. Biochem., 2008). In another report, sorghum protein wascross-linked with dextran or galactomannan with the aid of TGAse inorder to get a stable complex (E. Babiker and A. Kato, MolecularNutrition & Food Research).

The present disclosure provides methodologies utilizing the enzyme TGaseto attach the biocompatible polymer POZ and POZ derivatives tobiopharmaceuticals, such as but not limited to, proteins andpolypeptides. As discussed above, one advantage of POZ is that thehydrophilicity of the polymer can be varied by changing the nature ofthe R₇ group, such as, but not limited to, changing an alkyl grouppresent in the R₇ position from methyl to ethyl to propyl; these changescan lead to differences in the PK profile and to greater activity asshown by an increase in the area under the activity curve.

A potential concern relating to the producing desiredPOZ-biopharmaceutical conjugates is that POZ amines may make poor TGasesubstrates because of steric hindrance resulting from the side chainspresent in POZ. TGase catalyzes acyl transfer between the γ-carboxyamidegroup of the glutamine (acyl donor) and the polymeric primary amine(acyl acceptor), and it is reasonable to hypothesize that side chains onthe polymer would lead to steric inhibition of the acyl transfer. Theinventors of the present disclosure have found that simple POZ aminesare quite inefficient at TGase-mediated protein conjugation. The presentdisclosure reveals that moving the terminal amine group farther from thePOZ side chains by use of spacer moieties can lead to a practicalprocess giving efficient coupling of POZ amines to biopharmaceuticals,such as but not limited to, proteins and polypeptides. In oneembodiment, the POZ derivative contains an amine group separated fromthe POZ polymer by a spacer moiety of 3, 4, 5, 6, 7 8, 9 or 10 or moreatoms, such as, but not limited to, carbon, oxygen, nitrogen and sulfuratoms.

The structure of the products obtained from this reaction can berepresented by the formula:

Polypeptide-CH₂CH₂—CO—NH-POZ

In this structure the —CH₂CH₂—CO— group represents a protein glutamineresidue. Specific embodiments of the POZ amines falling under thegeneral formula above include, but are not limited to:

-   -   CH₃—[N(COCH₂CH₃)CH₂CH₂]_(n)—OCO—NH—CH₂CH₂—NH₂    -   CH₃—[N(COCH₂CH₃)CH₂CH₂]_(n)—S—S—CH₂CH₂—CO—NH—CH₂CH₂—NH₂    -   CH₃—[N(COCH₂CH₃)CH₂CH₂]_(n)—S—S—CH₂CH₂—CO—NH—(CH₂)₅—NH₂    -   CH₃—[N(COCH₂CH₃)CH₂CH₂]_(n)—S—S—CH₂CH₂—CO—NH—(CH₂)₆—NH₂        As discussed herein, the nature of the group at R₇ and the        initiator position can be varied, with ethyl and methyl being        used for exemplary purposes only

POZ-Lipid Conjugates

The present disclosure also provides for POZ-lipid conjugates. POZ-lipidconjugates of the present disclosure comprise a lipid portion linked toa polyoxazoline portion; the polyoxazoline portion may be any suchpolyoxazoline polymer of derivative described or referenced herein. Inone embodiment, the lipid portion comprises at least one hydrophobicmoiety and a chemical group capable of forming a linkage with a chemicalgroup on the polyoxazoline portion. In an alternate embodiment, thelipid portion comprises two hydrophobic moieties and the chemical groupis located at the head group position and the polyoxazoline portion islinked to the lipid portion through the chemical group located at headgroup position.

Lipids are a class of molecules that contain a hydrophobic portion and ahydrophilic portion. The hydrophobic and hydrophilic portions provide anamphipathic property to these molecules allowing them to aggregate in aspecific manner to form bilayers and vesicles/liposomes in aqueousenvironments. Phospholipids are a type of lipids that have suchamphipathic character. The head group of a phospholipid is hydrophilicwhereas the tail groups are hydrophobic. The hydrophilic head groupcontains the negatively charged phosphate group, and may contain otherpolar groups. The hydrophobic tail group generally comprises long fattyacid hydrocarbon chains. When placed in an aqueous environment,phospholipids form a variety of structures depending on the specificproperties of the phospholipid.

The lipid portion of the polyoxazoline-lipid conjugates may comprise anylipid capable of forming a vesicle/liposome, either alone or incombination with other lipid components of the liposomal compositions(described below). The lipids may be synthetic or naturally occurring.Regardless of the exact nature of the lipid comprising the lipidportion, the lipid contains a chemical group that is suitable forforming a linkage with a chemical group on the polyoxazoline portion.The nature of the linkage will depend on the chemical group present onthe polyoxazoline portion and the chemical group present on the lipidportion. In one embodiment, the chemical group that forms the linkagewith the polyoxazoline portion is located in the head group of the lipidportion. For example, the chemical group may be an amine group, hydroxylgroup, aldehyde group or a carboxylic acid group; other chemical groupsare not excluded. The polyoxazoline portion may be conjugated viaappropriate chemical group on the initiator or the terminal end of thepolymer.

In general the covalent attachment of polymers to a vesicle-forminglipid is accomplished by reaction of an active chemical group on thepolyoxazoline portion with a complementary chemical group on the lipidportion. The chemical groups on the polyoxazoline portion and/or thelipid portion may be activated prior to the reaction (such as, but notlimited to, removal of any protecting groups). A hydroxyl, amine orcarboxyl group may be activated for coupling by monofunctionalactivating agents, such as N-hydroxysuccinimide, ethylchloroformate,DCCD, Woodward's Reagent K, cyanuric acid and trifluoromethanesulfonylchloride among others. A number of bifunctional crosslinking reagentscontaining groups with different reactivities, such as somediisocyanates, may also be used.

The hydrophobic moieties are typically acyl chains containing an alkylportion. The alkyl portion of the acyl chain may vary in length; inaddition the alkyl portion may be saturated (contain no double bonds) orcontain one or more areas of unsaturation (contain one or more doublebonds). When unsaturated, the alkyl portion may have varying degrees ofunsaturation, for example, from 1 to 4 areas of unsaturation. When thealkyl portion contains an area of unsaturation, the hydrogen atoms atthe double bond may be in the cis or trans configuration. In oneembodiment, the alkyl portion of the acyl chains contains from 14 to 24carbons. When the lipid portion contains two hydrophobic moieties, thealkyl portions of the two hydrophobic moieties may be the same or may bedifferent.

Exemplary lipids, POZ-lipid conjugates, methods of synthesizing the sameand methods of using the foregoing are described in InternationalApplication No. PCT/US09/50286, which is hereby incorporated byreference for such teachings.

Liposomal Compositions

The present disclosure also provides for liposomal compositions. TheLiposomal compositions of the present disclosure incorporate apolyoxazoline-lipid conjugates of the present disclosure and provide anumber of advantages over liposomal compositions of the prior art. Forexample, the liposomal compositions of the present disclosure provide alonger residence time for the liposomal composition in the body; as suchthe liposomal compositions can release entrapped target molecules, suchas a therapeutic agent, over a longer period of time. In addition,prolonged residence times allow the liposomal composition to effectivelyreach various sites in the body and enter such regions.

The polyoxazoline-lipid conjugate of the present disclosure is used inpreparing a liposomal composition. In one embodiment, the liposomalcomposition contains a therapeutic agent for the treatment of humandisease. In an alternate embodiment, the liposomal composition containsa diagnostic agent. In still a further embodiment, the liposomalcomposition contains a targeting agent to target the liposomalcomposition to a particular cell or tissue. Liposomal compositions ofthe present disclosure may also contain combinations of the foregoing(for example, a therapeutic agent and a targeting reagent or adiagnostic agent and a targeting agent). In one embodiment thepolyoxazoline-lipid conjugate when incorporated in a liposomalcomposition is present at a mole ratio of about 0.5% to about 50% molepercent in the lipid layer of the liposomal composition, at a mole ratioof about 1% to about 30% mole percent in the lipid layer of theliposomal composition, at a mole ratio of about 2% to about 20% molepercent in the lipid layer of the liposomal composition or at a moleratio of about 5% to about 10% mole percent in the lipid layer of theliposomal composition. In such embodiment, the polyoxazoline-lipidconjugate may form a layer which is effective to extend the bloodcirculation time of the liposomes over that of the liposomes lacking thepolyoxazoline-lipid conjugate.

The liposomal composition comprises a polyoxazoline-lipid conjugate ofthe present disclosure in combination with other lipid components(lacking polyoxazoline components) that are capable of forming vesiclesand/or liposomes (the lipid components lacking a polyoxazoline componentare referred to as underivatized lipids). The underivatized lipidsinclude any amphipathic lipids having hydrophobic and polar head groupmoieties, and which (a) can form spontaneously into bilayer vesicles inwater or (b) are stably incorporated into lipid bilayers, with itshydrophobic moiety in contact with the interior, hydrophobic region ofthe bilayer membrane, and its polar head group moiety oriented towardthe exterior, polar surface of the membrane.

Exemplary liposomal compositions containing POZ-lipid conjugates,methods of synthesizing the same and methods of using the foregoing aredescribed in International Application No. PCT/US09/50286, which ishereby incorporated by reference for such teachings.

Target Molecule-POZ Conjugates

The present disclosure describes a variety of monofunctional POZderivatives capable of forming a linkage with a target molecule toproduce a hydrolytically stable target molecule-POZ conjugate asdiscussed above. In a general embodiment, the present disclosureprovides for a hydrolytically stable target molecule-POZ conjugatehaving the general formula (IV):

A-B-TM  (IV)

Wherein,

A is a monofunctional POZ derivative described herein, minus any leavinggroups eliminated during the reaction of the active functional group onthe POZ derivative with a binding partner on the target molecule;

-   -   TM is a target molecule; and    -   B is a hydrolytically stable linkage formed between the active        functional groups of a monofunctional POZ derivative of the        present disclosure and a binding partner on the target molecule,        it being understood that the nature of the hydrolytically stable        B linkage will depend on the nature of the active functional        group on the monofunctional POZ derivative and the binding        partner on the target molecule. Exemplary active functional        groups, binding partners and B linkages are provided in Table 3        below. The listing in Table 3 is not meant to be exhaustive and        other combinations and resulting B linkages may be envisioned        given the teachings of the present disclosure.

As discussed above, the POZ component used in the formation of thePOZ-target molecule conjugate is a POZ derivative as described inInternational Patent Application Nos. PCT/US2008/078159,PCT/US2009/030762 or PCT/US2009/050286.

TABLE 3 Active functional Binding Partner group on target Molecule Blinkage Tresylate SH Thioether (—S—) Maleimide SH Thioether (—S—) Activecarbonate NH₂ Urethane (—NH—CO—O—) Active ester NH₂ Amide (—NH—CO—)Aldehyde NH₂ (amine) Amine (—NH—)

GENERAL EXAMPLES

Examples 1-36 of U.S. patent application Ser. No. 12/529,001 are herebyincorporated by reference.

Materials and General Methods

Reagents were purchased from EM Science, Acros Organics, ABCR orAldrich. Dry solvents were prepared by distillation followed by dryingby distillation over calcium hydride. Monomers were distilled overcalcium hydride or freeze-dried using dry benzene. GPC was performed onan Agilent Technologies instrument with an RI detector. Two Phenogel GPCcolumns (Phenomenex, 5 microns, 300×7.8 mm) were used in series at 60°C.). The mobile phase was DMF. A calibration curve was generated withM-PEOZ-OH samples of different molecular weights as determined by MALDITOF. MALDI-TOF MS was performed with a Bruker Microflex with dithranolmatrix. The samples were prepared by mixing chloroform solutions of thepolymer and matrix (10 mg/mL) in a ratio of 1:1 (v/v). NMR spectra wererecorded in CDCl₃ on a Varian 500 MHz instrument.

Example 1 Preparation of 20 kDa H-PEOZ-Hydrazide

The synthesis of 20 kDa polyoxazoline succimidyl thiopropionate has beendescribed (H-PEOZ-T-SPA 20 K, GFC shows 83% of —O—Su; GPC showed aMn=18,243 Da, PDI=1.055, Mp=19,973 Da; and MALDI-TOF showed a Mn=20,972Da). Hydrazine monohydrate, NH₂NH₂; ·H₂O, 98% was from Aldrich, FW50.06, d 1.032.

H-PEOZ-T-SPA 20 K (2.0 gm, 9.1240×10⁻⁵ mol, 1 equiv.) was firstdissolved in anhydrous dichloromethane (90 mL) and this solution wastransferred into an addition funnel. In a 250 mL round bottom flask,hydrazine monohydrate (452 μL, 9.1240×10⁻³ mol, 100 equiv.) wasdissolved in anhydrous dichloromethane (5 mL). Under an argonatmosphere, the H-PEOZ-T-SPA solution in dichloromethane was added tothe hydrazine solution drop wise over one hour with rapid stirring. Thissolution was stirred overnight at ambient temperature and in an argonatmosphere. The white precipitate that was formed in the reactionmixture was filtered out and the filtrate was collected and concentratedto near dryness on a rotary evaporator. The remaining highlyconcentrated solution was further dried under vacuum for one hour. Theresidual solid was next dissolved in deionized water (40 mL) and sodiumchloride (6 gm) was added to the solution. The pH of the solution wasadjusted to 3.0 by the slow addition of 1.0 N HCl acid. This solutionwas next extracted three times with dichloromethane (50 mL each time).The collected dichloromethane pool was dried over anhydrous sodiumsulfate, filtered, and concentrated with a rotary evaporator and thenprecipitated in ethyl ether. The precipitated solid was recovered byfiltration, and was dried under vacuum. Yield: 1.8 gm. HPLC analysisshowed a 97% of substitution of H-PEOZ-Hydrazide (data not shown).

Example 2

Conjugation of Heparin by 20 kDa H-PEOZ-Hydrazide through CarbohydrateReducing Terminus

A 50 mg/mL solution of 20 kDa H- PEOZ-Hydrazide (2,222 μL, 5.5556×10⁻⁶mole, 5 equiv.) was added to heparin sodium salt (Grade I-A, purchasedfrom Sigma-Aldrich, MW 17000−19000 Da, 20 mg, 1.1111×10⁻⁶ mole, 1equiv.) in pH 3.0 acetic acid solution. The solution was allowed to stirat room temperature for 10 minutes. The solution pH was adjusted to 3.0by adding 0.1 N HCl acid. To this solution, freshly prepared 1 M NaBH₃CNin deionized water (111.1 μL, 1.1111×10⁻⁴ mole, 100 equiv) was added.The solution was stirred at room temperature for 10 minutes, and thenincubated at 37° C. for overnight. The reaction mixture was analyzed byGFC using a BioSEP-SEC-S 4000 column. GFC showed the formation ofH-PEOZ-heparin conjugate (data not shown).

Example 3

Conjugation of Dynorphin A (1-13) at C-terminus by 5 kDaM-PEOZ-Hydrazide using EDC as Coupling Agent

Dynorphin A (dynorphin 1-13) (Bachem, 0.55 mg, 2.2133×10⁻⁷ mol, 1equiv.) was dissolved in 250 μL of 50 mM 2-(N-morpholino)ethanesulfonicacid (MES) buffer at pH 3.0. A 109.5 μL aliquot of 50 mg/mL 5 kDaM-PEOZ-Hydrazide 5K (5.5 mg, 1.1066×10⁻⁶ mol, 5 equiv) in 50 mM MESbuffer at pH 3.0 was prepared and filtered through a 0.2 μm syringefilter. This 5 kDa M-PEOZ-Hydrazide solution was then added to thedynorphin A solution. The solution pH was adjusted to 3.0 by drop bydrop addition of a 50 mM HCl acid solution. A solution ofN-(3-Dimethylaminopropyl)-N′-ethyl-carbodiimide hydrochloride (EDC,purchased from Fluka) in deionized water (31 μL, 100 mg/mL, 1.6097×10⁻⁵mol, 20 equiv.) was added into the mixture. The solution pH was adjustedto 3.0 by slowly adding 50 mM HCl. The solution was allowed to stir atroom temperature for 3 hours. The reaction mixture was analyzed bySDS-PAGE, which confirmed the formation of mono-PEOZ-Dynorphin Aconjugate. The reaction mixture of 5 kDa M-PEOZ-Hydrazide and DynorphinA (1-13) was analyzed by SDS-PAGE using an XCell SureLock Mini-CellElectrophoresis System. A 4-12% NuPAGE Bis-Tris Mini Gel was used with1× MES SDS Running Buffer. The electrophoresis was done for 35 minuteswith constant voltage at 200 V. The reaction mixture (10 μL) waspre-mixed with 10 pL NuPAGE LDS Sample Buffer (4×) and 20 μL ofdeionized water. The gel was stained by GelCode Blue Stain, andde-stained. Lane 1: Mark 12 unstained protein standard. 10 μL. Lane 2:M-PEOZ-Dynorphin A conjugation mixture after 3 hours of reaction, 10 μLloading.

The POZ 5 K conjugate showed in-vitroμ-, δ-, κ-opioid binding activitysimilar to that of unconjugated dynorphin A (1-13).

Example 4

Conjugation of Lysozyme at Carboxylate Groups by 5 kDa M-PEOZ-Hydrazideusing EDC as Coupling Agent

Lysozyme (from chicken egg white, MW 14,307 Da, purchased fromSigma-Aldrich, 26 mg, 1.63995×10⁻⁶ mol, 1 equiv.) was dissolved in 1 mLof 50 mM (2-(N-morpholino)ethanesulfonic acid (MES) buffer at pH 3.0. Analiquot of 50 mg/mL 5 kDa M-PEOZ-Hydrazide (Mn 4,952 Da, 812 μL,8.1998×10⁻⁶ mol, 5 equiv) in 50 mM MES buffer at pH 3.0 was prepared andfiltered through a 0.2 μm syringe filter. The 5 kDa M-PEOZ-Hydrazidesolution was added to the lysozyme solution. The solution pH wasadjusted to 3.0 by addition of 0.1 N HCl. An aliquot of a freshlyprepared 100 mg/mL solution ofN-(3-Dimethylaminopropyl)-N′-ethyl-carbodiimide hydrochloride (EDC,purchased from Fluka) in DI water (64 μL, 3.2799×10⁻⁵ mol, 20 equiv.)was added into the mixture. The solution pH was adjusted to 3.0 byaddition of 0.1 N HCl. The solution was allowed to stir at roomtemperature for 1 hour, and then kept frozen at −20° C. The solution wasanalyzed by reversed phase HPLC and SDS-PAGE. Both reversed phase HPLCand SDS-PAGE continued the formation of mono-, di-, tri- and quad- 5 kDaPEOZ-Lysozyme conjugates. Reversed phase HPLC was performed with aWaters Symmetry SB300 C4 Column using a mobile phase of 0.1% TFA inwater (A) and 0.1% TFA in ACN (B). A linear gradient of mobile phaseswere used during run. Flow rate at 0.5 mL/min; UV at 280 nm.

Example 5 Synthesis of H-PEOZ-O—NH₂5 kDa

-   -   a. Synthesis of 5 kDa H-PEOZ-O—NH(Boc)

Hydroxybenzotriazole (HOBT, 0.0655 g, 0.485 mmoles) was dissolved in 10mL of acetonitrile and dried by azeotropic evaporation using a rotaryevaporator. Boc-aminooxy acetic acid (0.0445 g, 0.233 mmoles) was addedand the mixture was dissolved in 10 mL of dichloromethane.Dicyclohexylcarbodiimide (DCC) (0.060 g, 0.291 mmoles) was added as asolid and the mixture was stirred at room temperature for 2 hours.H-PEOZ-thioamine (M_(n)=5,150 Da, 1.0 g, 0.194 mmoles) was added and themixing was continued overnight. The next day the mixture was filteredusing a 0.2 μm syringe filter and precipitated by slow addition intodiethyl ether. The supernatant ether layer was decanted and the residuewas collected and dried. The resulting white powder was dried undervacuum to give 1.02 g of the desired compound with a yield of 99%.

¹H NMR (Varian, 500 MHz, 10 mg/mL CDCl₃) spectrum showed the usualbackbone peaks at 1.12 ppm (m, 3H, CH₃CH₂CO—); 2.31 ppm (m) and 2.41 (s)(total area 2H, CH₃CH₂CO—); and 3.46 ppm (m, 4H, —NCH₂CH₂N—). Theterminal group peaks appear at 1.48 ppm (s, 9H, —CH₂ONHBoc), 2.73 ppm(m, 4H, —CH₂SCH₂CH₂NHBoc), and 4.32 ppm (br s, 2H, —CH₂ONHBoc).

-   -   b. Synthesis of 5 kDa H-PEOZ-O—NH₂

H-PEOZ-O—NH-Boc (1.02 g, 0.192 mmoles) was dissolved in methanolic HCl(3N in MeOH, 20 mL) and stirred for 40 min at room temperature. Themixture was concentrated and dried using a rotary evaporator. Theresidue was dissolved in dichloromethane and precipitated by slowaddition into diethyl ether. The supernatant ether layer was decantedand the residue was collected and dried. The resulting white powder wasdried under vacuum to give 1.00 g of the desired compound with a yieldof ˜100%.

¹H NMR (Varian, 500 MHz, 10 mg/mL CDCl₃) spectrum showed the usualbackbone peaks at 1.14 ppm (m, 3H, CH₃CH₂CO—); 2.32 ppm (m) and 2.41 (s)(total area 2H, CH₃CH₂CO—); and 3.47 ppm (m, 4H, -NCH₂CH₂N—). Theterminal group peak was found at 2.72 ppm (m, 4H, —CH₂SCH₂CH₂NH—), 4.16ppm (m, 2H, —SCH₂CH₂NH—CO—CH₂—ONH₂). The deprotection of Boc group wasalso confirmed by the disappearance of the peak at 1.48 ppm (s, 9H,—CH₂ONHBoc). GPC gave Mp 5182 Da and PD of 1.06.

The aminooxy end group was characterized via derivatization with4-methoxy benzaldehyde. H-PEOZ-ONH2 (0.0688 g, 0.0133 mmoles), wasreacted overnight with 4-methoxy benzaldehyde (32.0 0.263 mmoles) in 5mL of 0.1 M acetate buffer (counterion sodium, pH 3.6). The mixture wasextracted using dichloromethane, concentrated using a rotary evaporator,and precipitated by slow addition into diethyl ether. The supernatantether layer was decanted and the residue was collected and dried. Theresulting white powder was dried under vacuum to give 0.070 g of thedesired compound with a yield of −100%.

¹H NMR (Varian, 500 MHz, 10 mg/mL CDCl₃) spectrum showed the usualbackbone peaks at 1.14 ppm (m, 3H, CH₃CH₂CO—); 2.32 ppm (m) and 2.41 (s)(total area 2H, CH₃CH₂CO—); and 3.47 ppm (m, 4H, —NCH₂CH₂N—). Thederivatized terminal group peaks were found at 2.67 ppm (m, 4H,—CH₂SCH₂CH₂NH—), 3.79 ppm (s, 3H, —Ar—OCH₃), 4.42 ppm (m, 2H,—SCH₂CH₂NH—CO—CH₂—O—), 6.86 ppm (d, 2H, aromatic), 7.48 ppm (m, 2H,aromatic), 8.11 ppm (d, 1H, —ON═CH—Ar).

Example 6

Coupling of H—PEOZ-T—NH₂ (1%) to Hyaluronic acid (HA)

Sodium hyaluronate (HA, Mw 6×10⁵, 100 mg, 0.167 mop and H-PEOZ-T-NH₂ (Mn4,381 Da, 11.1 mg, 2.54 μmol) were dissolved in 50 mM2-(N-morpholino)ethanesulfonic acid (MES) buffer (7 mL) and the pH wasadjusted to 4.5-5.0. After addition of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, 2.4 mg, 12.7 μmol), the solution wasstirred for 3 hours at room temperature. The reaction was stopped by theaddition of 1 M HCl solution and the pH was adjusted to 2.0. Thesolution was stirred for another 30 minutes. The aqueous solution wasthen washed with dichloromethane (3×10 mL) to remove any unreactedH-PEOZ-T-NH₂. The resulting aqueous solution was lyophilized to collecta white powder.

The degree of PEOZ conjugated to HA is verified by ¹H-NMR. It wasdetermined that 1.03% of the available carboxylic acids were coupledwith PEOZ. This was done by comparing relative integrations of the NMRpeaks of —NHCOCH₃ on HA and —COCH₂CH₃ on PEOZ backbone.

Example 7 Preparation of 5 kDa M-PEOZ-ethylenediamine.

M-PEOZ-p-nitrophenylchlorofonnate 5 kDa was prepared as per procedurespreviously described, and dissolved in dry chloroform. Ethylenediaminewas added and pH adjusted to approximately 8.0 withN-ethyldiisopropylamine (DEAP). The mixture was maintained atroom-temperature overnight, and then purified by repeated extractionswith a KHSO₄ buffer. The organic layer was separated, dried withanhydrous Na₂SO₄ and concentrated under vacuo. Ethyl ether was added andthe resulting precipitate was collected by filtration and then dried.The content of amine-terminated side chains was determined by Snyderassay and ¹H-NMR.

Example 8 Conjugation of 5 kDa M-PEOZ-ethylenediamine to GCSF Catalyzedby TGAse

The conjugation of POZ-amines to the glutamine residues on a protein iscatalyzed by the enzyme transglutaminase (TGAse).

A solution containing granulocyte colony stimulating factor (GCSF, 1.5mg/mL) in 10 mM phosphate buffer pH 7.0 and M-PEOZ-NH₂ (5 kDa) at 10fold molar excess with respect to the protein, was prepared. Aftercomplete dissolution of the polymer and the protein, TGAse was added atan enzyme to substrate ratio (E/S) of 1:75 by weight and the reactionmixture was incubated at room temperature for 4 h.

In order to compare the reaction rates, PEG-NH₂ (5 kDa) was conjugatedto GCSF using the same reaction conditions as above.

Under the conditions described, there was no completion of conjugationbetween G-CSF and PEOZ-NH₂, while conjugation between PEG and G-CSF waspartially complete. Addition of 2 mg of TGAse enzyme to the reactionmixture catalyzed the conjugation process and allowed for the formationof POZ-GCSF. Maldi-TOF mass analysis confirmed the product to have amolecular weight of 23.04 kDa corresponding to the molecular weight of amonoconjugated POZ-GCSF.

The results above show that the rate of conjugation of GCSF with POZ isslower than conjugation of GCSF with PEG. While not being limited byalternate explanations, it is possible the slower reaction rate isattributable to steric hindrance from the adjacent side chains presenton the POZ backbone.

Example 9 Preparation of 10 kDa PEOZ-Ethylenediamine andPEOZ-Cadaverine. Conjugation of PEOZ-SPA (NHS) to Ethylenediamine

After dissolution of ethylenediamine in 10 mL of anhydrousdichloromethane, previously prepared 10 kDa PEOZ-succimidyl propionate(SPA, an NHS ester) was added in small increments and the reactionmixture was then allowed to stir for 18 h. After washing with 0.1 N HClin order to remove any low molecular weight compounds, the organic phasewas separated and dried with anhydrous sodium sulfate, and thenconcentrated under vacuo. The residue was redissolved in water anddialyzed against water for 2 days. The content of amine-terminated sidechains was verified by Snyder assay and ¹H-NMR.

Conjugation of PEOZ-SPA (NHS) to Cadaverine

The linkage of a longer amine spacer to PEOZ-COOH was carried out underthe same conditions reported for the conjugation to ethylendiamine.

Example 10 Conjugation of 10 kDa PEOZ-Ethylenediamine and 10 kDaPEOZ-Cadaverine to GCSF Catalyzed by TGAse

To a solution containing GCSF (1.5 mg/mL) in 10 mM phosphate buffer pH7.0, 10 kDa PEOZ-ethylenediamine (PEOZ-Et) or 10 kDa PEOZ-cadaverine(PEOZ-Cad) at 10 fold molar excess with respect to the protein, wasadded. TGAse was added at an enzyme to substrate ratio (E/S) of 1:75 byweight and the reaction mixtures were incubated at room temperature for4 h. The reactions were monitored by RP-HPLC using a Zorbax Cis column(4.6×250mm; Agilent, USA) and eluting with a linear gradient of 40-70%acetonitrile containing 0.05% TFA and over a 25 min run time, followedby an isocratic wash at 80% acetonitrile containing 0.05% TFA. Theeffluent from the column was monitored by measuring the absorbance at280 nm. For the PEOZ-Et, conjugate yield was 30% or less (data notshown). For PEOZ-Cad conjugate yield was essentially complete at 98%(98% of conjugate, less than 2% of native protein) (data not shown).

The reaction mixture was purified by ion exchange chromatography usingan analytical strong-cation exchange column (Tosoh-Haas, SP-SPW,7.5mm×7.5cm). The column was pre-equilibrated with 10 mM phosphatebuffer pH 4.7 at a flow rate of 1 mL/min and the protein conjugate waseluted with a NaCl gradient (0.01 to 0.1 M) over 90 min. The producteluent fraction (monitored by the UV absorbance at 280 nm) wascollected, dialyzed against 10 mM acetate buffer pH 4.0 to remove anylow molecular weight impurities and stored at 4° C. Identification ofthe reaction mixtures and the final purified products was verified bySDS gel electrophoresis.

Example 11 Conjugation of 20 kDa PEOZ-ethylenediamine and 20 kDaPEOZ-cadaverine to G-CSF Catalyzed by TGAse

20 kDa PEOZ- ethylenediamine (PEOZ-Et) and 20 kDa PEOZ-cadaverine(PEOZ-Cad) were prepared starting from 20 kDa PEOZ-SPA (NHS) under thesame conditions reported in

Example 9

The obtained amine polymers without further purification were linked toGCSF by TGase catalysis. The reaction was carried out under the sameconditions as reported above in Example 10.

Example 12 Conjugation of 20 kDa PEOZ-Hexylamine to GCSF Catalyzed byTGAse

20 kDa PEOZ-hexylamine was prepared starting from 20 kDa PEOZ-SPA (NHS)under the same conditions reported above for the preparation of 1- kDaPEOZ-NH2. To a solution of GCSF (12.6mg, 1 equiv.) in 2 mL was added a50 mg/mL solution of H-PEOZ-Hex-NH2 (MW 20 kDa, 5 equiv.) in 10 mMsodium phosphate buffer at pH 7.0, followed by addition of TGAse (33.6mg of Activa RM contains 1 wt % Transglutaminase, obtained fromAjinomoto Food Ingredient LLC). The reaction mixture was allowed toshake gently at room temperature for 5 hours, and then acidified to 4.0with IN HCl. The crude mixture was analyzed by SEC-HPLC using aPhenomenex BioSEP SEC 53000 column, and monitored at 280 nm. The mobilephase contained 50 mM sodium phosphate buffer at pH 6.4 with 5 v/v %ethanol. SEC-HPLC showed about 65% of PEOZ conjugation, predominantlymono-conjugated species (data not shown).

The solution was loaded onto a 60.5 mL SP Sepharose HP 26/114 column(media from GE Healthcare and packed in-house) pre-equilibrated with 20mM sodium acetate buffer at pH 4.0. Bound proteins were step eluted with20 mM sodium acetate buffer at pH 4.0, 1 M NaCl. Fractions containingthe 20 kDa H-PEOZ-Hex-GCSF were pooled, concentrated, and bufferexchanged into 10 mM sodium acetate buffer at pH 4.0 with 0.004 v/v %Tween 20 by ultrafiltration in an Amicon Stirred Ultrafiltration Cell.The conjugate was analyzed by SEC-HPLC, which showed thatH-PEOZ-20K-Hex-GCSF conjugate was free from native G-CSF anddi-PEOZ-G-CSF.

Example 13 Conjugation of 10 kDa and 20 kDa PEOZ-aldehyde at theN-terminus of GCSF by Reductive Amination

A solution of 62.94 mg/mL H-PEOZ-Propionaldehyde (1.8 mL, 10 equiv. forMW 20 kDa) in 1 mM HCl was added into GCSF solution (1.0 equiv of 0.6mg/mL in sorbital, Tween 20, and acetate buffer). The solution pH was4.6. Following five minutes of shaking at room temperature, a freshlyprepared solution of sodium cyanoborohydride (NaBH₃CN) in deionizedwater (200 μL, 850 mM) was added to the mixture. The solution wasallowed to shake gently at 4° C. for 21 hours. The solution pH wasadjusted to 4.0 by 0.1 N HCl. The crude reaction mixture was analyzed bySEC-HPLC using a Phenomenex BioSEP SEC 53000 column, and monitored at280 nm. The SEC-HPLC showed 51% PEOZ conjugation.

The mixture was purified on a 60 mL SP Sepharose HP column (mediapurchased from GE Healthcare; the column was packed in-house)pre-equilibrated with 20 mM sodium acetate buffer at pH 4.0. Boundproteins were step eluted with 20 mM sodium acetate buffer at pH 4.0containing 1 M NaCl. Fractions containing the H-PEOZ-N^(ter)-GCSF werepooled, concentrated, and buffer exchanged into 10 mM sodium acetatebuffer at pH 4.0 with 0.004 v/v % Tween 20 by ultrafiltration in anAmicon™ Stirred Ultrafiltration Cell (Millipore, Model 8050, 50 mL)using a regenerated cellulose membrane (Millipore, YM10, Dia. 44.5 mm,NMWL 10,000). The purity of purified H-PEOZ-20 K-N^(ter)-GCSF wasanalyzed by SEC-HPLC and shows the conjugate consisted of >99% of monoH-PEOZ-N^(ter)-G-CSF. The results were confirmed by and SDS-PAGE (datanot shown).

The results of the conjugation yield from examples 10, 11, 12 and 13 aresummarized in Table 4 below.

TABLE 4 Degree of PEOZ-GCSF conjugation Conjugation degree Amount ofnative Conjugates (%) protein (%) 20 kDa M-PEOZ-Et-GCSF 20 80 20 kDaM-PEOZ-Cad-GCSF 95 5 20 kDa H-PEOZ-Hex-GCSF 65-75* 25-35* 20 kDaH-PEOZ-N^(ter)-GCSF 51 49 10 kDa M-PEOZ-Et-GCSF 30 70 10 kDaM-PEOZ-Cad-GCSF 98 2 10 kDa H-PEOZ-N^(ter)-GCSF 49 51*range from different batches prepared on different scales.

The results above show that the longer NH₂ spacer on the PEOZ allows fora more complete conjugation. PEOZ-Et-NH₂ was less reactive than eitherPEOZ-Cad-NH, or PEOZ-Hex-NH₂ with respect to conjugation yields andamount of native GCSF. The foregoing data surprisingly show that theamine-polymer with ethylenediamine (Et) as a spacer, for any PEOZmolecular weight, is a poorer substrate when compared to the aminepolymer with cadaverine (Cad) or hexylamine (Hex) as the spacer.

Example 14

Determination of Potency of PEOZ-GCSF Conjugates by NFS 60 cellProliferation Assay

The NFS-60 cell lines are murine myeloblastic cells established fromleukemic cells obtained after infection of (NFS X DBA/2) F1 adult micewith Cas Br-M murine leukemia virus. The cells respond to murineinterleukin (IL)-3 (IL3), murine or human IL4, human IL6,granulocyte/macrophage colony stimulating factor (GMCSF), GCSF anderythropoietin. The promyelocytic state of NFS-60 cells is maintained inthe presence of GCSF. The cells differentiate into neutrophils andmacrophages in the presence of IL3 and GMCSF.

These cells were used to measure the in-vitro activity of the PEOZ-GCSFcompounds (10 kDa and 20 kDa H-PEOZ-N^(ter)-GCSF; and 20 kDaH-PEOZ-GCSF). Sample dilutions of the test articles were made in cellmedia containing no growth factors. The concentrations prepared were0.12, 0.024, 0.049, 0.097, 0.195, 0.39, 0.78, 1.56, 3.10, 6.25, 12.5 and25.0 ng/mL. 96-well plates seeded with NFS 60 cells at a cell density of2,500 cells/well. The 96-well plates were incubated at 37° C. in anO₂/CO₂ atmosphere. Test wells received a dilution of the test compoundsin cell media (minus growth factors); negative control wells received anequal volume of cell media (minus growth factors); positive controlsreceived an equal amount of GCSF (unconjugated) diluted in an equalvolume of cell media (minus growth factors). At 24, 48 and 72 hours cellnumber was determined using an optical absorbance assay. WST-1 solutionwas then added to each well, and after a 1-2 hour incubation period theabsorbance of each solution was measured at 450 and 620nm. The amount offormazan dye color produced is a reflection of the viability and numberof the cells in each well. The optical density (450-620 nm) is thenplotted against GCSF concentration (ng/mL) and compared against thecontrol.

The results of this study are portrayed in FIG. 4 and show the threeconjugates of PEOZ-GSCF have similar activity to that of unconjugatedGCSF. The EC50 activity (using the Hill-Slope method) of GCSF, 10 kDaPEOZ-N^(ter)-GCSF, 20 kDa PEOZ-N^(ter)-GCSF, 20 kDa PEOZ-hex-GCSF wereabout 0.06, 0.08, 0.1 and 0.12 ng/mL, respectively.

Example 15 Evaluation the In-Vivo Activity of Polymeric GCSF.

Sprague-Dawley rats (male, 7-8 weeks old) were used in this study with 5animals per test article group. The animals were allowed at least 4 daysfor acclimatization and were allowed food and water ad libitum. Theanimals were dosed on Day 0, intravenously through the tail vein witheither G-CSF, 10 kDa PEOZ-N^(ter)-GCSF, 20 kDa PEOZ-N^(ter)-GCSF, 20 kDaPEOZ-hex-GCSF. The dose was 100 μg of protein/kg of animal weight. Bloodwas collected at the following time points: pre-dose, 6, 12, 24, 48, 72,96, and 120 h post-dose. At each time point, approximately 0.6 mL ofblood was removed via a jugular vein catheter with the aid of a syringe.An aliquot of approximately 0.25 mL was placed in tubes containinganticoagulant (K₂EDTA) for CBC analysis and stored at 4° C. Theremaining blood sample was placed in serum separator tubes and used toharvest serum. Serum was collected, transferred to labeled tubes, andstored at −70° C. FIG. 5 illustrates the blood neutrophil counts in maleSprague-Dawley rats following intravenous injection of GCSF and 10 kDaPEOZ-N^(ter)-GCSF, 20 kDa PEOZ-N^(ter)-GCSF, 20 kDa PEOZ-hex-GCSF (100μg/kg; n=5, ±SEM).

Example 16 Conjugation of 5 kDa POZ-Ethylenediamine to hGH Catalyzed byTGAse

A solution containing human growth hormone (hGH; 1.5 mg/mL) in 10 mMphosphate buffer pH 7.0 and POZ-NH₂ (5 kDa) at 10 fold molar excess withrespect to the protein, was prepared. After complete dissolution of thepolymer and the protein, TGAse was added at an enzyme to substrate (E/S)ratio of 1:75 by weight and the reaction mixture was incubated at roomtemperature for 4 h. The reaction mixture was analyzed on a ZorbaxGF-250 column (4.6×250 mm) eluting at a flow rate of 0.3 mL/min using0.2 M phosphate buffer pH 7+20% acetonitrile. The eluent from the columnwas monitored by measuring the absorbance at 280 nm. Gel filtrationchromatography and SDS-page electrophoresis showed the presence of amono- and a di-conjugate of hGH; approximately 20% of the native hGH wasstill present resulting in a conjugation efficiency of 80%.

Example 17

Conjugation of 5 kDa POZ-Ethylenediamine to Apomyoglobin (apoMb)Catalyzed by TGAse

A solution containing apoMb (0.7 mg/mL) in 10 mM phosphate buffer pH 7.0and POZ-NH₂ (5 kDa) at 100 fold molar excess with respect to theprotein, was prepared. After complete dissolution of the polymer and theprotein, TGAse was added at an enzyme to substrate (E/S) ratio of 1:75by weight and the reaction mixture was incubated at room temperature for4 h. The reaction mixture was analyzed on a Zorbax C18 column (4.6×250mm) flushing at a flow rate of 1 mL/min. The eluent from the column wasmonitored by measuring the absorbance at 280 nm. The separation wasachieved by a linear gradient of 40-70% acetonitrile containing 0.05%TFA over a 25 min period, followed by an isocratic wash at 80%acetonitrile containing 0.05% TFA. The product was retained and theresults are shown in FIG. 6.

Example 18 Conjugation of 30 kDa PEOZ-NHS to Erythropoietin (EPO)

A 10 mL solution of 1.326 mg/mL EPO solution (13.26 mg, 1.0 equiv.) wasprepared; the pH of the solution was adjusted to 8.0 by 0.1 N NaOH. Tothe EPO solution a 1084 μL aliquot of 25 mg/mL H-PEOZ-T-SPA (30 kDa; MW33,293 Da, 27.1 mg, 1.5 equiv.) in 2 mM HCl was added. The solution wasallowed to stir gently at room temperature, while its pH was maintainedat 7.9-8.2 by 0.1 N NaOH. Following 45 min of reaction, the reaction wasacidified to pH 3.0 with 0.5 M citric acid. Using an AKTA Purifiersystem (GE Healthcare/Amersham Biosciences), the acidified mixture wasloaded onto a 10 mL SP Sepharose HP column (two 5 mL columns connectedin series) pre-equilibrated with 20 mM sodium citrate buffer at pH 3.0.Bound proteins were step eluted with 20 mM sodium citrate buffer at pH3.0 containing 1 M NaCl. Fractions that contain mono-H-PEOZ-EPO werepooled, neutralized to pH 6.9 by 0.1 N NaOH, concentrated, and bufferexchanged into a pH 6.9 formation buffer, which contains phosphate,citrate, sodium chloride, by ultrafiltration in an Amicon™ StirredUltrafiltration Cell (Millipore, Model 8050, 50 mL) using a regeneratedcellulose membrane (Millipore, YM10, Dia. 44.5 mm, NMWL 10,000). Thefinal conjugate was analyzed by SEC-HPLC with a Shodex Protein KW-803column monitored at 280 nm using formulation buffer as mobile phase;results were confirmed by SDS-PAGE (data not shown). MALDI-TOF analysisof the purified final product shows the Mn of the conjugate was 59,850.4Da.

A PEOZ-EPO conjugate with 40 kDa H-PEOZ-T-SPA was also made with thesame procedure as described above.

Example 19 Identification of Sites of Polymer Conjugation

Peptide mapping of digested native EPO and PEOZ conjugated EPO wasconducted to determine which amino acid residues were modified afterconjugation with PEOZ-NHS. In the procedure, mono PEOZ-EPO (30 kDa and40 kDa) and unconjugated EPO were digested with Endoprotease Lys C, andthen mapped on a reverse phase C-18 chromatography column to identifythe modified versus unmodified peptide segments as described below.

-   -   a. Denaturation and reduction:

Aliquots of mono PEOZ-EPO solution (347 μg/mL) and unconjugated EPOsolution (1.326 mg/mL) were first denatured in a solution containingguanidine HCl (6 M) and EDTA (6 mM) in a pH 8.0 300 mM NaPO₄ buffercontaining DTT (1.5 μmol). Each aliquot solution contained 100 μg of EPOcontent. These solutions were gently mixed overnight at roomtemperature.

-   -   b. Carboxymethylation:

Monoiodoacetic acid (1.2 mg of 3.15 μmol) was added to each solution andthe vials were covered with aluminum foil, to avoid light exposure, andthe mixed room temperature for 45 minutes. The solutions were injectedinto dialysis cartridges (NMW 2000) and dialyzed against 50 mM Tris HClbuffer (pH8.5)

-   -   c. Digestion:

The EPO solution was removed from the dialysis cartridge and placed inreaction tubes. Endoprotease Lys-C enzyme (10 μg) was dissolved in 300mM NaPO₄ solution (100 μL). 20 μL of this Lys-C solution was added tothe EPO solution and allowed to digest overnight at 37° C. The next day,the digestion was quenched with 10% TFA solution ( 1/10^(th) the volumeof the digestion solution).

-   -   d. Peptide mapping:

Each of the quenched digested solution was analyzed on a C-18 reversephase column (Jupiter 5u 300 A 250 mm×4.6 mm) using gradient flow of0.1% TFA in H₂O and 0.1% TFA in ACN mobile phase with a flow rate of 1.0mL/min. The analytes were detected at a wavelength of 220 nM.

FIGS. 7 and 8 are chromatograms of the peptide maps of Endoprotease LysC digested EPO and PEOZ-EPO, respectively. The digestion of EPO yields 9peptide fragments, of which 8 fragments are easily chromatographicallyseparated: L1 (AA 1-20), L2 (AA 21-45), L3 (AA 46-52), L4 (AA 53-97), L5(AA 98-116), L6 (AA 117-140), L7 (AA 141-152) and L9 (AA 155-165). L8(AA 153-154) has two amino acids and was not retained on thechromatography column. The pattern of digested EPO is similar to thatreferenced in previous publications. In the chromatograms for theconjugated PEOZ-EPO samples, the L3 & L6 peaks decrease significantly inrelation to the L7 peak suggesting that conjugation primarily occurs inthe L3 segment at Lysine 52. In addition, the L1 peak also reduces inrelation to L7. This suggests that PEOZ is primarily conjugated atLysine 52 and secondarily to Alanine 1. A new broad peak is detectedbetween the L5 and L4 peaks which correspond to a monoconjugated PEOZ 30kDa peptide fragment of the L3-L4 segment and the LI segment. Note thatPEOZ has an absorbance at 220 nm unlike other polymers such as PEG.

Example 20 Determination of Potency of PEOZ-EPO Conjugates by UT-7 CellProliferation Study

The UT-7 human leukemia cell line is used to measure EPO dependent cellproliferation activity. UT-7 cells were used to measure the in-vitroactivity of the PEOZ-EPO onjugates (30 K and 40 K H-PEOZ-EPO). Dilutionsof the test articles and control were made in cell media. Theconcentrations prepared were 0.125 to 2500 ng/mL. 96 well plates wereseeded at a cell density of 4×10⁵ cells per mL. The 96-well plates wereincubated at 37° C. in an O₂/CO₂ atmosphere for 4 days. Test wellsreceived a dilution of the test compounds in cell media (minus growthfactors); negative control wells received an equal volume of cell media(minus growth factors); positive controls received an equal amount ofEPO (unconjugated) diluted in an equal volume of cell media (minusgrowth factors). At 24, 48 and 72 hours cell number was determined usingan optical absorbance assay. WST-1 solution was then added to each well,and after a 1-2 hour incubation period the absorbance of each solutionwas measured at 450 and 620 nm. The amount of formazan dye colorproduced is a reflection of the viability and number of the cells ineach well. The optical density (450-620 nm) is then plotted against EPOconcentration (ng/mL) and compared against the control.

The results of this study are portrayed in FIG. 9 and they show that twoconjugates of PEOZ-EPO (30 kDa and 40 kDa) have similar activity to thatof unconjugated EPO. The EC₅₀ of native EPO, H-PEOZ-EPO 30 kDa andH-PEOZ-EPO 40 kDa was 4.23 ng/mL, 10.9 ng/mL and 8.8 ng/mL,respectively.

Example 21 In-Vivo Pharmacokinetics and Pharmacodynamics

15 male Sprague-Dawley Rats, 7-8 weeks old, were used in this study. Theanimals were allowed at least 4 days for acclimatization and wereallowed food and water ad libitum. The 15 animals were divided intothree groups of 5 animals each. Each group of animals was dosed at Day 0intravenously (through tail vein) with one dose of either EPO orPEOZ-EPO (30 kDa); control animals received a sham injection. The dosewas 25 μg of protein/kg of animal weight. Blood samples were taken atthe following time points: pre-dose on day 0 and post-dose at days 1, 3,7, 11, and 15.

The sample was divided into 2 aliquots. One aliquot was used to measurefor reticulocyte counts, hemoglobin levels, RBC, platelets, usingmicrocell counter. The second aliquot was used to prepare serum forbioanalytical measurements. Serum was collected into tubes containing K₂EDTA as the anticoagulant. The bioassay employs the quantitativesandwich enzyme immunoassay technique. A pre-coated plate specific forhuman EPO was blocked for at least one hour and washed. Standards andsamples were pipetted into the wells and any human EPO present was boundby the immobilized antibody. After washing away any unbound substances,a sulfo-tag linked antibody was added to the wells. Following a wash toremove any unbound antibody detection reagent, 1× read buffer was addedto the wells and the plate is read immediately byelectrochemiluminescence detection (ECL) which emits light in proportionto the concentration. The assay ranges for EPO and PEOZ-EPO were 96 to70,000 pg/mL and 576 to 420,000 pg/mL, respectively.

FIG. 10 is a pharmacokinetic profile that shows the concentrations ofEPO and a PEOZ-EPO conjugate in rat serum over a period of 15 days. Thedata shows PEOZ extends the residence of EPO in rat by a factor of tentimes.

FIG. 11 is a pharmacodynamic profile that shows the effect of EPO andPEOZ-EPO conjugates on the reticulocyte counts in rat blood over aperiod of 15 days. The data shows that reticulocyte counts are increasedby a factor of 1.5 times even at day 7.

The foregoing description illustrates and describes certain embodimentsof the compounds and applications of the present disclosure.Additionally, the disclosure shows and describes only the exemplaryembodiments of the compounds and applications, but as mentioned above,it is to be understood that the teachings of the present disclosure arecapable of use in various other combinations, modifications, andenvironments and is capable of changes or modifications within the scopeof the concepts as expressed herein, commensurate with the aboveteachings and/or the skill or knowledge of the relevant art. Theembodiments described hereinabove are further intended to explain bestmodes known of practicing the invention and to enable others skilled inthe art to utilize the invention in such, or other, embodiments and withthe various modifications required by the particular applications oruses of the invention. Accordingly, the description is not intended tolimit the invention to the form disclosed herein. All references citedherein are incorporated by reference as if fully set forth in thisdisclosure.

What is claimed:
 1. A terminally activated polyoxazoline (POZ) compoundof the general structure R₁-POZ-P_(p)-Q_(q)-W_(w)-U_(u)-X, wherein POZis [N(COR₇)CH₂CH₂]_(n) X is an active functional group; P is a linkinggroup selected from the group consisting of: —O—, —S—, NH, —NR₁₁—,unsubstituted heterocyclyl, Q is a linking group selected from the groupconsisting of: unsubstituted or substituted alkyl, unsubstituted orsubstituted alkenyl, unsubstituted or unsubstituted aralkyl,unsubstituted or substituted heterocyclyl, unsubstituted or substitutedaryl, —(CH₂)_(m)—CONH—(CH₂)_(m)—, —NH—(CH₂)_(m)—NHCO—(CH₂)_(m)—,—CO—(CH₂)_(m)—, —CO—C₆H₄—, —CO—R₈—, —(R₁₅)_(m)— or —(CR₃R₄)_(m)—; U is alinking group selected from the group consisting of: —(R₁₆)_(o)—,—(CR₅R₆), —NH—R₂₁—NHCO—R₂₂—; W is a linking group selected from thegroup consisting of: —NH—CO—O—, —NH—CO, —NH—CO—NH—, —NH—CS—NH—, —O—,—S—, —NH— and SO—CO—NH; R₇ is independently selected for each repeatingunit of POZ from an unsubstituted or substituted alkyl, alkenyl oraralkyl group; R₁, R₃-R₆, R₁₁, R₁₅-R₁₆ and R₂₁-R₂₂ are eachindependently selected from hydrogen, unsubstituted or substitutedalkyl, alkenyl or aralkyl group; R₈ is —C₆H₁₀—CH₂—; n is an integer from3 to 1000; m and o are integer independently selected from 1 to 10; p,q, w and u are integers independently selected from zero or one.
 2. Theterminally activated POZ compound of claim 1, wherein the activefunctional group is protected.
 3. The terminally activated POZ compoundof claim 1, wherein R₇ is an unsubstituted or substituted alkyl, alkenylhaving from 1 to 12 carbon atoms.
 4. The terminally activated POZcompound of claim 1, wherein R₇ is methyl, ethyl or n-propyl.
 5. Theterminally activated POZ compound of claim 1, wherein the activefunctional group is selected from the group consisting of: aldehyde,active carbonate (—O—CO—Z), maleimide, sulfonate ester (OSO₂—R₂₃),hydrazide, epoxide, iodoacetamide, alkyne, azide, isocyanate, cyanateisothiocyanate, thiocyanate, nitrile, a carbonyldiimidazole derivative,vinylsulfone, carboxylic acid halide, active ester (—CO—Z) andcarboxylic acid, wherein any of the foregoing may be substituted orunsubstituted, Z is an activating group and R₂₃ is an unsubstituted orsubstituted alkyl, alkenyl, alkynyl, aralkyl or aryl group.
 6. Theterminally activated POZ compound of claim 5, wherein the sulfonateester is tresylate or mesylate.
 7. The terminally activated POZ compoundof claim 5, wherein Z is N-succinimidyloxy, chlorine, bromine,sulfo-N-succinimidyloxy, p-nitrophenoxy, 1-imidazolyl,or1-benzotriazolyloxy.
 8. The terminally activated POZ compound of claim5, wherein the heterocycle of the active functional group is asubstituted or unsubstituted piperazinyl or a substituted piperidinylgroup.
 9. The terminally activated POZ compound of claim 1, wherein thePOZ polymer has a polydispersity value of less than or equal to 1.05.10. The terminally activated POZ of claim 1 linked to a target molecule.11. The terminally activated POZ compound of claim 1, wherein the activefunctional group forms a linkage with a target molecule and wherein alllinkages between the target molecule and the POZ compound arehydrolytically stable in a biological system.
 12. The terminallyactivated POZ of claim 1, wherein p is
 1. 13. The terminally activatedPOZ of claim 1, wherein p is 1, q is 0 or 1, and one of w or u is
 0. 14.A terminally activated POZ of claim 1, wherein p is 1, q is 0, and w andu are
 1. 15. A compound having a structure:

wherein, n is an integer from 3 to 1000.